Nanoparticle formulations

ABSTRACT

This present disclosure relates to methods and compositions comprising biologically active nanoparticle formulations of MYC protein. Provided are methods of making the nanoparticle formulations and methods of using the nanoparticle formulations for treatment.

PRIORITY

This application is a division of U.S. patent application Ser. No.15/828,971, filed Dec. 1, 2017, which claims priority to U.S.Provisional Application Ser. No. 62/429,466, filed Dec. 2, 2016, thecontent of each of which is incorporated by reference in its entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Jan. 12, 2020, isnamed 106417-0451_SL.txt and is 39,185 bytes in size.

BACKGROUND

The quaternary structure of polypeptides can greatly influence theirphysicochemical and biological character. Protein aggregation, ornon-native aggregation, refers to the process by which protein moleculesassemble into stable complexes composed of two or more proteins, withthe individual proteins denoted as the monomer. Aggregates are oftenheld together by strong non-covalent contacts, and require some degreeof conformational distortion (unfolding or misfolding) in order topresent key stretches of amino acids that form the strong contactsbetween monomers. While aggregation tends to increase the stability ofprotein, it often does so at the cost of biological activity of theprotein, decreased uniformity of the composition, and can, in somecases, increase the immunogenicity of the protein. These propertiesadversely affect the ability to use such proteins as a biologic fortreatment.

SUMMARY

The present technology relates to the controlled assembly ofMYC-containing polypeptides into populations of biologically activeparticles of defined size range. Methods are provided herein for theproduction of stable preparations of MYC-containing nanoparticles thatretain biologic activity. Also provided are methods using thebiologically active particles for treatment, including in vitro and invivo methods of treating cells.

Disclosed herein are compositions comprising MYC-containing polypeptidesformulated as biologically active, stable nanoparticles. In someembodiments, the MYC-containing polypeptides comprise a fusion peptide,wherein the fusion peptide comprises: (i) a protein transduction domain;(ii) a MYC polypeptide sequence, and wherein the nanoparticles exhibitthe biological activity of MYC. In some embodiments, the fusion peptidecomprises SEQ ID NO: 1. In some embodiments, the fusion peptidecomprises SEQ ID NO: 10.

Provided herein, in certain embodiments, are compositions comprising apopulation of biologically active nanoparticles comprising one or moreMYC-containing polypeptides. In some embodiments, the number averagediameter of the biologically active nanoparticles is between about 80 nmand about 150 nm. In some embodiments, pH of the formulation is at leastabout pH 6.0, but is no greater than about pH 8. In some embodiments,contacting an anti-CD3 or anti-CD28 activated T-cell with theMYC-containing polypeptide nanoparticle composition under conditionssuitable for T-cell proliferation, augments one or more of theactivation, survival, or proliferation of the T-cell compared with ananti-CD3 or anti-CD28 activated T-cell that is not contacted with theMYC polypeptide-containing composition. In some embodiments, the MYCpolypeptide is acetylated. In some embodiments, the MYC-containingpolypeptide comprises a MYC fusion peptide, comprising a proteintransduction domain linked to a MYC polypeptide. In some embodiments,the MYC fusion peptide further comprises one or more molecules that linkthe protein transduction domain and the MYC polypeptide. In someembodiments, the MYC-containing polypeptide comprises a MYC fusionpeptide with the following general structure: protein transductiondomain-X-MYC sequence, wherein -X- is molecule that links the proteintransduction domain and the MYC sequence. In some embodiments, theprotein transduction domain sequence is a TAT protein transductiondomain sequence. In some embodiments, the TAT protein transductiondomain sequence is selected from the group consisting of TAT[48-57] andTAT[57-48]. In some embodiments, the MYC polypeptide is a MYC fusionpeptide comprising SEQ ID NO: 1. In some embodiments, the MYCpolypeptide is a MYC fusion peptide comprising SEQ ID NO: 10. In someembodiments, the nanoparticles have a number average diameter of betweenabout 80 nm and about 150 nm. In some embodiments, the nanoparticleshave a number average diameter of between about 100 nm and about 110 nm.In some embodiments, the composition further comprises, apharmaceutically acceptable excipient. In some embodiments, thecomposition is formulated for topical administration, oraladministration, parenteral administration, intranasal administration,buccal administration, rectal administration, or transdermaladministration.

Also provided herein, in certain embodiments, are methods of increasingone or more of activation, survival, or proliferation of one or moreimmune cells or increasing an immune response in a subject in needthereof by administering a therapeutically effective amount of acomposition provided herein comprising a population of biologicallyactive nanoparticles comprising one or more MYC-containing polypeptides.In some embodiments, the one or more immune cells comprise one or moreanergic immune cells. In some embodiments, the one or more immune cellsare T cells. In some embodiments, the T cells are selected from thegroup consisting of naïve T cells, CD4+ T cells, CD8+ T cells, memory Tcells, activated T cells, anergic T cells, tolerant T cells, chimeric Bcells, and antigen-specific T cells. In some embodiments, the one ormore immune cells are B cells. In some embodiments, the B cells areselected from the group consisting of naïve B cells, plasma B cells,activated B cells, memory B cells, anergic B cells, tolerant B cells,chimeric B cells, and antigen-specific B cells. Also provided herein, incertain embodiments, are methods of priming hematopoietic stem cells toenhance engraftment, following hematopoietic stem cell transplantation(HSCT) comprising, contacting one or more hematopoietic stem cells, invitro, with the composition provided herein comprising a population ofbiologically active nanoparticles comprising one or more MYC-containingpolypeptides prior to transplantation of the hematopoietic stem cells.

Also provided herein, in certain embodiments, are methods for thepreparation of a population of biologically active nanoparticlescomprising one or more MYC-containing polypeptides, the methodcomprising: (a) solubilizing MYC-containing polypeptides in asolubilization solution comprising a concentration of a denaturing agentto provide solubilized MYC-containing polypeptides; (b) performing afirst refolding step on the solubilized MYC-containing polypeptides witha first refold buffer comprising about 0.35 to about 0.65 theconcentration of the denaturing agent of step (a) and about 100 mM toabout 1M alkali metal salt and/or alkaline metal salt for at least about30 to 180 minutes to provide a first polypeptide mixture; (c) performinga second refolding step on the first polypeptide mixture with a secondrefold buffer comprising about 0.10 to about 0.30 the concentration ofthe denaturing agent of step (b) and about 100 mM to 1M alkali metalsalt and/or alkaline metal salt at least about 30 to 180 minutes toprovide a second polypeptide mixture; (e) performing a third refoldingstep on the second polypeptide mixture with a third refold buffercomprising about 100 mM to 1M alkali metal salt and/or alkaline metalsalt for at least about 30 to 180 minutes; and (f) maintaining theMYC-containing polypeptides in the third refold buffer for a period oftime sufficient to produce biologically active nanoparticles having anumber average diameter of between about 80 nm and about 150 nm, whereincontacting an anti-CD3 or anti-CD28 activated T-cell with thebiologically active nanoparticles under conditions suitable for T-cellproliferation, augments one or more of the activation, survival, orproliferation of the T-cell compared with an anti-CD3 or anti-CD28activated T-cell that is not contacted with the biologically activenanoparticles. In some embodiments, the first refolding step, secondrefolding step, and/or third refolding step comprise performing the stepby buffer exchange. In some embodiments, buffer exchange is performedusing tangential flow filtration. In some embodiments, the alkali metalsalt comprises one more of a sodium salt, a lithium salt, and apotassium salt. In some embodiments, the alkali metal salt comprises oneor more of sodium chloride (NaCl), sodium bromide, sodium bisulfate,sodium sulfate, sodium bicarbonate, sodium carbonate, lithium chloride,lithium bromide, lithium bisulfate, lithium sulfate, lithiumbicarbonate, lithium carbonate, potassium chloride, potassium bromide,potassium bisulfate, potassium sulfate, potassium bicarbonate, andpotassium carbonate. In some embodiments, the alkaline salt comprisesone more of a magnesium salt and a calcium salt. In some embodiments,the alkaline metal salt comprises one or more of magnesium chloride,magnesium bromide, magnesium bisulfate, magnesium sulfate, magnesiumbicarbonate, magnesium carbonate, calcium chloride, calcium bromide,calcium bisulfate, calcium sulfate, calcium bicarbonate, and calciumcarbonate. In some embodiments, the alkali metal salt comprises sodiumchloride (NaCl). In some embodiments, the first, second, and/or thirdrefold buffers comprise about 500 mM NaCl. In some embodiments, theconcentration of denaturing agent in step (a) is from about 1 M to about10 M. In some embodiments, the denaturing agent comprises one or more ofguanidine, guanidine hydrochloride, guanidine chloride, guanidinethiocyanate, urea, thiourea, lithium perchlorate, magnesium chloride,phenol, betain, sarcosine, carbamoyl sarcosine, taurine,dimethylsulfoxide (DMSO); alcohols such as propanol, butanol andethanol; detergents, such as sodium dodecyl sulfate (SDS), N-lauroylsarcosine, Zwittergents, non-detergent sulfobetains (NDSB), TRITONX-100, NONIDET™ P-40, the TWEEN™ series and BRIJ™ series; hydroxidessuch as sodium and potassium hydroxide. In some embodiments, the firstrefold buffer, the second refold buffer, and/or third refold buffer eachindependently comprise a buffering agent. In some embodiments, thebuffering agent comprises one or more of TRIS(Tris[hydroxymethyl]aminomethane), HEPPS(N-[2-Hydroxyethyl]piperazine-N′-[3-propane-sulfonic acid]), CAP SO(3-[Cyclohexylamino]-2-hydroxy-1-propanesulfonic acid), AMP(2-Amino-2-methyl-1-propanol), CAPS(3-[Cyclohexylamino]-1-propanesulfonic acid), CHES(2-[N-Cyclohexylamino]ethanesulfonic acid), arginine, lysine, and sodiumborate. In some embodiments, the buffering agent is independentlypresent at a concentration from about 1 mM to about 1M. In someembodiments, the first refold buffer, second refold buffer, and/or thirdrefold buffer each independently comprise an oxidizing agent and areducing agent, wherein a mole ratio of oxidizing reagent to reducingagent is from about 2:1 to about 20:1. In some embodiments, theoxidizing agent comprises cysteine, glutathione disulfide (“oxidizedglutathione”), or both. In some embodiments, the oxidizing agent isincluded in a concentration from about 0.1 mM to about 10 mM. In someembodiments, the reducing agent comprises one or more ofbeta-mercaptoethanol (BME), dithiothreitol (DTT), dithioerythritol(DTE), tris(2-carboxyethyl)phosphine, (TCEP), cystine, cysteamine,thioglycolate, glutathione, and sodium borohydride. In some embodiments,the reducing agent is included in a concentration from about 0.02 mM toabout 2 mM. In some embodiments, the denaturing agent comprises urea. Insome embodiments, the denaturing agent comprises 6-8M urea. In someembodiments, the first, second, and/or third refold buffers compriseglutathione and/or oxidized glutathione. In some embodiments, the first,second, and/or third refold buffers comprise 5 mM glutathione and/or 1mM oxidized glutathione. In some embodiments, the first, second, and/orthird refold buffers comprise glycerol. In some embodiments, the step(f) is performed for at least 5 hours. In some embodiments, the step (f)is performed for at least 10 hours. In some embodiments, the step (f) isperformed for 10-12 hours. In some embodiments, the step (f) furthercomprises stirring the MYC-containing polypeptides in the third refoldbuffer at less than 1000 rpm.

In some embodiments, the methods provided herein further compriseisolating a recombinant MYC-containing polypeptide from a microbial hostcell. In some embodiments, the microbial host cell is E. coli. In someembodiments, isolating a recombinant MYC-containing polypeptide from amicrobial host cell comprises expressing the MYC-containing polypeptidefrom an inducible promoter. In some embodiments, isolating a recombinantMYC-containing polypeptide from a microbial host cell comprisespurifying the MYC-containing polypeptide using affinity chromatographyand/or anion exchange chromatography. In some embodiments, theMYC-containing polypeptide is acetylated.

In some embodiments, the MYC-containing polypeptides of the nanoparticlecompositions and methods for the production thereof provided herein arerecombinant polypeptides. In some embodiments, the MYC-containingpolypeptide of the nanoparticle compositions provided herein comprises aMYC fusion peptide, comprising a protein transduction domain linked to aMYC polypeptide. In some embodiments, the MYC fusion peptide furthercomprises one or more molecules that link the protein transductiondomain and the MYC polypeptide. In some embodiments, the MYC-containingpolypeptide comprises a MYC fusion peptide with the following generalstructure: protein transduction domain-X-MYC sequence, wherein -X- ismolecule that links the protein transduction domain and the MYCsequence. In some embodiments, protein transduction domain sequence is aTAT protein transduction domain sequence. In some embodiments, TATprotein transduction domain sequence is selected from the groupconsisting of TAT[48-57] and TAT[57-48]. In some embodiments, theMYC-containing polypeptide is a MYC fusion peptide comprising SEQ IDNO: 1. In some embodiments, the MYC-containing polypeptide is a MYCfusion peptide comprising SEQ ID NO: 10. In some embodiments, thenanoparticles have a number average diameter from about 80 nm and about150 nm. In some embodiments, the nanoparticles have a number averagediameter from about 100 nm and about 110 nm.

In an exemplary embodiment, provided herein is a method for thepreparation of a population of biologically active nanoparticlescomprising one or more MYC-containing polypeptides, the methodcomprising: (a) denaturing MYC-containing polypeptides in a bufferedsolubilization solution comprising 6-8M Urea to provide denaturedMYC-containing polypeptides; (b) performing a first refolding step onthe denatured MYC-containing polypeptides with a first refold buffercomprising about 3M Urea and about 500 mM NaCl for at least about 120minutes to provide a first polypeptide mixture; (c) performing a secondrefolding step on the first polypeptide mixture by buffer exchange witha second refold buffer comprising about 1.5M Urea and about 500 mM NaClat least about 120 minutes to provide a second polypeptide mixture; (d)performing a third refolding step on the second polypeptide mixture bybuffer exchange with a third refold buffer comprising about 500 mM NaClfor at least about 120 minutes; and (f) maintaining the MYC-containingpolypeptides in the third refold buffer for a period of time sufficientto produce biologically active nanoparticles having a number averagediameter of between about 80 nm and about 150 nm, wherein contacting ananti-CD3 or anti-CD28 activated T-cell with the biologically activenanoparticles under conditions suitable for T-cell proliferation,augments one or more of the activation, survival, or proliferation ofthe T-cell compared with an anti-CD3 or anti-CD28 activated T-cell thatis not contacted with the biologically active nanoparticles.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A and FIG. 1B show the effect of MYC-containing nanoparticleformulation C2A on proliferation of anti-CD3 and anti-CD28 activatedT-cells using flow cytometry techniques. FIG. 1A shows the proliferationof T-cells in the buffer control sample. FIG. 1B shows flow theproliferation of T-cells incubated for 24 h with MYC-containingnanoparticle formulation C2A.

FIG. 2A and FIG. 2B show chromatograms of select MYC-containingnanoparticle formulations analyzed by Asymmetrical Flow Field-flowFractionation (AF4) and Multiangle Laser Light Scattering (MALLS). FIG.2A and 2B show the chromatograms obtained from the analysis ofFormulation F01 and Formulation F02, respectively. Trace t1 and t2 wereobtained from samples stored at 25° C. Trace t2* and t4* were obtainedfrom samples stored at 5° C. Time (t) was measured in weeks and is shownin the numerical value that follows the t (i.e., t1=one week, t2=twoweeks etc.).

FIG. 3 shows a comparison of chromatograms derived by fractionatingbiologically active and inactive MYC-containing nanoparticleformulations by size exclusion chromatography (SEC) using Sepax SRT-C2000 and 300 columns in tandem. Traces showing fractionation ofbiologically active nanoparticle formulations C12 and C13 and thebiologically inactive formulation R147 are indicated.

FIG. 4A and FIG. 4B show analysis of MYC-containing nanoparticleformulation C2A using Size Exclusion Chromatography with Multi-AngleLight Scattering analysis (SEC-MALS). FIG. 4A shows retention volume(ml) as a function of refractive index (left ordinate, RV×RI) ormolecular size (right ordinate, RV×MW). FIG. 4B shows chromatogramsshowing fractionation of biologically active MYC-containing nanoparticleformulations and a biologically inactive formulation.

FIG. 5A-D depicts size distribution of MYC-containing nanoparticles inselect formulations analyzed by Dynamic Light Scattering (DLS)technique. FIG. 5A depicts size distribution of MYC-containingnanoparticle formulation C2A by DLS. FIG. 5B depicts size distributionof non-biologically active MYC-containing nanoparticle formulation R149tested at a concentration of 0.5 mg/ml. FIG. 5C depicts the nanoparticlesize distribution of biologically active MYC-containing nanoparticleformulation C12 tested at a concentration of 0.5 mg/ml. FIG. 5D showsthe nanoparticle size distribution of biologically active MYC-containingnanoparticle formulation C13 tested at a concentration of 0.5 mg/ml.

FIG. 6A and FIG. 6B shows analysis of MYC-containing nanoparticleformulation C2A using Nanoparticle Tracking Analysis (NTA) technique.FIG. 6A shows the tracings observed from triplicate determination ofnanoparticle formulation C2A. FIG. 6B shows the consensus tracing of thetriplicate determinations derived from the analysis of C2A nanoparticleformulation shown in FIG. 6A.

FIG. 7 shows analysis of MYC-containing nanoparticle formulation C2Busing electron microscopy technique.

FIG. 8 shows a comparison of Asp-N endoproteinase digests ofbiologically active and inactive MYC-containing nanoparticleformulations by peptide mapping analysis technique. Biologically activeMYC-containing nanoparticle formulations C2B, C6 and C7 (“functional”)were compared to biologically inactive MYC-containing nanoparticleformulations C4, 147 and 149 (“non-functional”).

FIG. 9 shows a representative peptide map for MYC-containingnanoparticle formulation C13 with the individual peptide peaksidentified.

FIG. 10A-C shows RP-HPLC Chromatograms for nanoparticle formulation C13.FIG. 10A shows the full chromatogram. FIG. 10B and FIG. 10C show twodifferent zoom views of full chromatogram in FIG. 10A.

FIG. 11 shows the results for Far UV CD Spectroscopy analysis ofnanoparticle formulation C13 depicted as mean residues molar ellipticity(deg cm² per decimole) as a function of wavelength (nm).

FIG. 12 shows the results for Near UV CD Spectroscopy analysis ofnanoparticle formulation C13 depicted as mean residues molar ellipticity(deg cm² per decimole) as a function of wavelength (nm).

FIG. 13 shows the Normalized Second Derivative Fourier transforminfrared spectra (FTIR) for four separate samples of nanoparticleformulation C14 in comparison with two control samples of bovine serumalbumin (BSA).

FIG. 14 shows Analytical Ultracentrifugation (AUC) data for nanoparticleformulation C13.

FIG. 15A and FIG. 15B show Non-reduced (NR) and Reduced (R) DenaturingSEC chromatograms, respectively, for nanoparticle formulation C13. Theoverlaid traces on each chromatogram represent testing that wasperformed approximately 1 month apart for samples stored at 2° C.-8° C.

FIG. 16A and FIG. 16B show denaturing SEC chromatograms for TAT-MYC andTAT-3AMYC nanoparticle formulations. FIG. 16B shows the chromatogram forTAT-MYC. TAT-MYC protein complex elutes between minutes 6 and 7. Smallerprotein multimers and excipient elute between minutes 8 and 15. FIG. 16Bshows the chromatograms for TAT-3AMYC compared to the functional TAT-MYCprotein preparations. The bulk of the non-functional TAT-3AMYC proteinpreparation is comprised of smaller protein multimers and excipientpeaks can be seen eluting between minute 8 and 17.

FIG. 17 shows the results of a T cell potency assay. TAT-MYC showed a 3fold increase the live population T-cell population compared to notreatment (NT). TAT-3AMYC showed no increase the live T-cell populationcompared to no treatment (NT).

FIG. 18 depicts size distribution of MYC-containing nanoparticles inselected formulations of Green Monkey TAT-MYC analyzed by Dynamic LightScattering (DLS) technique.

FIG. 19 shows RP-HPLC Chromatograms for Green Monkey TAT-MYC compared tohuman TAT-MYC.

FIG. 20 shows SEC-HPLC Chromatograms for Green Monkey TAT-MYC comparedto human TAT-MYC.

FIG. 21 shows the results of a T cell potency assay for Green MonkeyTAT-MYC compared to human TAT-MYC.

DETAILED DESCRIPTION

The present disclosure is not to be limited in terms of the particularembodiments described in this application, which are intended as singleillustrations of individual aspects of the disclosure. All the variousembodiments of the present disclosure will not be described herein. Manymodifications and variations of the disclosure can be made withoutdeparting from its spirit and scope, as will be apparent to thoseskilled in the art. Functionally equivalent methods and apparatuseswithin the scope of the disclosure, in addition to those enumeratedherein, will be apparent to those skilled in the art from the foregoingdescriptions. Such modifications and variations are intended to fallwithin the scope of the appended claims. The present disclosure is to belimited only by the terms of the appended claims, along with the fullscope of equivalents to which such claims are entitled.

It is to be understood that the present disclosure is not limited toparticular uses, methods, reagents, compounds, compositions orbiological systems, which can, of course, vary. It is also to beunderstood that the terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to belimiting.

In addition, where features or aspects of the disclosure are describedin terms of Markush groups, those skilled in the art will recognize thatthe disclosure is also thereby described in terms of any individualmember or subgroup of members of the Markush group.

As will be understood by one skilled in the art, for any and allpurposes, particularly in terms of providing a written description, allranges disclosed herein also encompass any and all possible subrangesand combinations of subranges thereof. Any listed range can be easilyrecognized as sufficiently describing and enabling the same range beingbroken down into at least equal halves, thirds, quarters, fifths,tenths, etc. As a non-limiting example, each range discussed herein canbe readily broken down into a lower third, middle third and upper third,etc. As will also be understood by one skilled in the art all languagesuch as “up to,” “at least,” “greater than,” “less than,” and the like,include the number recited and refer to ranges which can be subsequentlybroken down into subranges as discussed above. Finally, as will beunderstood by one skilled in the art, a range includes each individualmember. Thus, for example, a group having 1-3 cells refers to groupshaving 1, 2, or 3 cells. Similarly, a group having 1-5 cells refers togroups having 1, 2, 3, 4, or 5 cells, and so forth.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this disclosure belongs.

I. Definitions

The terminology used herein is for the purpose of describing particularembodiments only and is not intended to be limiting of the disclosure.As used herein, the singular forms “a”, “an” and “the” are intended toinclude the plural forms as well, unless the context clearly indicatesotherwise.

As used herein, the term “about” means that a value may vary +/−20%,+/−15%, +/−10% or +/−5% and remain within the scope of the presentdisclosure. For example, “a concentration of about 200 IU/mL”encompasses a concentration between 160 IU/mL and 240 IU/mL.

As used herein, the term “administration” of an agent to a subjectincludes any route of introducing or delivering the agent to a subjectto perform its intended function. Administration can be carried out byany suitable route, including intravenously, intramuscularly,intraperitoneally, or subcutaneously. Administration includesself-administration and the administration by another.

The term “amino acid” refers to naturally occurring and non-naturallyoccurring amino acids, as well as amino acid analogs and amino acidmimetics that function in a manner similar to the naturally occurringamino acids. Naturally encoded amino acids are the 20 common amino acids(alanine, arginine, asparagine, aspartic acid, cysteine, glutamine,glutamic acid, glycine, histidine, isoleucine, leucine, lysine,methionine, phenylalanine, proline, serine, threonine, tryptophan,tyrosine, and valine) and pyrolysine and selenocysteine. Amino acidanalogs refers to agents that have the same basic chemical structure asa naturally occurring amino acid, i.e., an α carbon that is bound to ahydrogen, a carboxyl group, an amino group, and an R group, such as,homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium. Such analogs have modified R groups (such as, norleucine) ormodified peptide backbones, but retain the same basic chemical structureas a naturally occurring amino acid. In some embodiments, amino acidsforming a polypeptide are in the D form. In some embodiments, the aminoacids forming a polypeptide are in the L form. In some embodiments, afirst plurality of amino acids forming a polypeptide are in the D formand a second plurality are in the L form.

Amino acids are referred to herein by either their commonly known threeletter symbols or by the one-letter symbols recommended by the IUPAC-IUBBiochemical Nomenclature Commission. Nucleotides, likewise, are referredto by their commonly accepted single-letter codes.

The terms “polypeptide,” “peptide,” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues. Theterms apply to naturally occurring amino acid polymers as well as aminoacid polymers in which one or more amino acid residues is anon-naturally occurring amino acid, e.g., an amino acid analog. Theterms encompass amino acid chains of any length, including full lengthproteins, wherein the amino acid residues are linked by covalent peptidebonds.

As used herein, a “control” is an alternative sample used in anexperiment for comparison purpose. A control can be “positive” or“negative.” For example, where the purpose of the experiment is todetermine a correlation of the efficacy of a therapeutic agent for thetreatment for a particular type of disease, a positive control (acomposition known to exhibit the desired therapeutic effect) and anegative control (a subject or a sample that does not receive thetherapy or receives a placebo) are typically employed.

As used herein, the term “effective amount” or “therapeuticallyeffective amount” refers to a quantity of an agent sufficient to achievea desired therapeutic effect. In the context of therapeuticapplications, the amount of a therapeutic peptide administered to thesubject may depend on the type and severity of the infection and on thecharacteristics of the individual, such as general health, age, sex,body weight and tolerance to drugs. It may also depend on the degree,severity and type of disease. The skilled artisan will be able todetermine appropriate dosages depending on these and other factors.

As used herein, the term “expression” refers to the process by whichpolynucleotides are transcribed into mRNA and/or the process by whichthe transcribed mRNA is subsequently being translated into peptides,polypeptides, or proteins. If the polynucleotide is derived from genomicDNA, expression may include splicing of the mRNA in a eukaryotic cell.The expression level of a gene may be determined by measuring the amountof mRNA or protein in a cell or tissue sample. In one aspect, theexpression level of a gene from one sample may be directly compared tothe expression level of that gene from a control or reference sample. Inanother aspect, the expression level of a gene from one sample may bedirectly compared to the expression level of that gene from the samesample following administration of the compositions disclosed herein.The term “expression” also refers to one or more of the followingevents: (1) production of an RNA template from a DNA sequence (e.g., bytranscription) within a cell; (2) processing of an RNA transcript (e.g.,by splicing, editing, 5′ cap formation, and/or 3′ end formation) withina cell; (3) translation of an RNA sequence into a polypeptide or proteinwithin a cell; (4) post-translational modification of a polypeptide orprotein within a cell; (5) presentation of a polypeptide or protein onthe cell surface; and (6) secretion or presentation or release of apolypeptide or protein from a cell.

The term “linker” refers to synthetic sequences (e.g., amino acidsequences) that connect or link two sequences, e.g., that link twopolypeptide domains. In some embodiments, the linker contains 1, 2, 3,4, 5, 6, 7, 8, 9, or 10 of amino acid sequences.

The terms “lyophilized,” “lyophilization” and the like as used hereinrefer to a process by which the material (e.g., nanoparticles) to bedried is first frozen and then the ice or frozen solvent is removed bysublimation in a vacuum environment. An excipient may be included inpre-lyophilized formulations to enhance stability of the lyophilizedproduct upon storage. The lyophilized sample may further containadditional excipients.

As used herein the term immune cell refers to any cell that plays a rolein the immune response. Immune cells are of hematopoietic origin, andinclude lymphocytes, such as B cells and T cells; natural killer cells;myeloid cells, such as monocytes, macrophages, dendritic cells,eosinophils, neutrophils, mast cells, basophils, and granulocytes.

The term “lymphocyte” refers to all immature, mature, undifferentiatedand differentiated white lymphocyte populations including tissuespecific and specialized varieties. It encompasses, by way ofnon-limiting example, B cells, T cells, NKT cells, and NK cells. In someembodiments, lymphocytes include all B cell lineages including pre-Bcells, progenitor B cells, early pro-B cells, late pro-B cells, largepre-B cells, small pre-B cells, immature B cells, mature B cells, plasmaB cells, memory B cells, B-1 cells, B-2 cells and anergic AN1/T3 cellpopulations.

As used herein, the term T-cell includes naïve T cells, CD4+ T cells,CD8+ T cells, memory T cells, activated T cells, anergic T cells,tolerant T cells, chimeric B cells, and antigen-specific T cells.

The term “B cell” or “B cells” refers to, by way of non-limitingexample, a pre-B cell, progenitor B cell, early pro-B cell, late pro-Bcell, large pre-B cell, small pre-B cell, immature B cell, mature Bcell, naïve B cells, plasma B cells, activated B cells, anergic B cells,tolerant B cells, chimeric B cells, antigen-specific B cells, memory Bcell, B-1 cell, B-2 cells and anergic AN1/T3 cell populations. In someembodiments, the term B cell includes a B cell that expresses animmunoglobulin heavy chain and/or light chain on its cells surface. Insome embodiments, the term B cell includes a B cell that expresses andsecretes an immunoglobulin heavy chain and/or light chain. In someembodiments, the term B cell includes a cell that binds an antigen onits cell-surface. In some embodiments disclosed herein, B cells orAN1/T3 cells are utilized in the processes described. In certainembodiments, such cells are optionally substituted with any animal cellsuitable for expressing, capable of expressing (e.g., inducibleexpression), or capable of being differentiated into a cell suitable forexpressing an antibody including, e.g., a hematopoietic stem cell, anaïve B cell, a B cell, a pre-B cell, a progenitor B cell, an earlyPro-B cell, a late pro-B cell, a large pre-B cell, a small pre-B cell,an immature B cell, a mature B cell, a plasma B cell, a memory B cell, aB-1 cell, a B-2 cell, an anergic B cell, or an anergic AN1/T3 cell.

The terms “MYC” and “MYC gene” are synonyms. They refer to a nucleicacid sequence that encodes a MYC polypeptide. A MYC gene comprises anucleotide sequence of at least 120 nucleotides that is at least 60% to100% identical or homologous, e.g., at least 60, 65%, 70%, 75%, 80%,85%, 86%, 87%, 88%, 90%, 91%, 92%, 94%, 95%, 96%, 97%, 98%, or any otherpercent from about 70% to about 100% identical to sequences of NCBIAccession Number NM_002467.5. In some embodiments, the MYC gene is aproto-oncogene. In certain instances, a MYC gene is found on chromosome8, at 8q24.21. In certain instances, a MYC gene begins at 128,816,862 bpfrom pter and ends at 128,822,856 bp from pter. In certain instances, aMYC gene is about 6 kb. In certain instances, a MYC gene encodes atleast eight separate mRNA sequences—5 alternatively spliced variants and3 unspliced variants.

The terms “MYC protein,” “MYC polypeptide,” and “MYC sequence” aresynonyms and refer to the polymer of amino acid residues disclosed inNCBI Accession Number NP_002458.2 (provided below) orUniProtKB/Swiss-Prot:P01106.1, which is human myc isoform 2, andfunctional homologs, variants, analogs or fragments thereof. Thissequence is shown below.

(SEQ ID NO: 2) MDFFRVVENQQPPATMPLNVSFTNRNYDLDYDSVQPYFYCDEEENFYQQQQQSELQPPAPSEDIWKKFELLPTPPLSPSRRSGLCSPSYVAVTPFSLRGDNDGGGGSFSTADQLEMVTELLGGDMVNQSFICDPDDETFIKNIIIQDCMWSGFSAAAKLVSEKLASYQAARKDSGSPNPARGHSVCSTSSLYLQDLSAAASECIDPSVVFPYPLNDSSSPKSCASQDSSAFSPSSDSLLSSTESSPQGSPEPLVLHEETPPTTSSDSEEEQEDEEEIDVVSVEKRQAPGKRSESGSPSAGGHSKPPHSPLVLKRCHVSTHQHNYAAPPSTRKDYPAAKRVKLDSVRVLRQISNNRKCTSPRSSDTEENVKRRTHNVLERQRRNELKRSFFALRDQIPELENNEKAPKVVILKKATAYILSVQAEEQKLISEEDLLRKRREQLKHKLEQLRNSCA

In some embodiments, the MYC polypeptide is a complete MYC polypeptidesequence. In some embodiments, the MYC polypeptide is a partial MYCpolypeptide sequence. In some embodiments, the MYC polypeptide is c-MYC.In some embodiments, the MYC polypeptide sequence comprises the sequenceshown below:

(SEQ ID NO: 3) MDFFRVVENQQPPATMPLNVSFTNRNYDLDYDSVQPYFYCDEEENFYQQQQQSELQPPAPSEDIWKKFELLPTPPLSPSRRSGLCSPSYVAVTPFSLRGDNDGGGGSFSTADQLEMVTELLGGDMVNQSFICDPDDETFIKNIIIQDCMWSGFSAAAKLVSEKLASYQAARKDSGSPNPARGHSVCSTSSLYLQDLSAAASECIDPSVVFPYPLNDSSSPKSCASQDSSAFSPSSDSLLSSTESSPQGSPEPLVLHEETPPTTSSDSEEEQEDEEEIDVVSVEKRQAPGKRSESGSPSAGGHSKPPHSPLVLKRCHVSTHQHNYAAPPSTRKDYPAAKRVKLDSVRVLRQISNNRKCTSPRSSDTEENVKRRTHNVLERQRRNELKRSFFALRDQIPELENNEKAPKVVILKKATAYILSVQAEEQKLISEEDLLRKRREQLKHKLEQLRKGELNSKLE.

In some embodiments, the MYC polypeptide sequence comprises the sequenceshown below:

(SEQ ID NO: 4) PLNVSFTNRNYDLDYDSVQPYFYCDEEENFYQQQQQSELQPPAPSEDIWKKFELLPTPPLSPSRRSGLCSPSYVAVTPFSLRGDNDGGGGSFSTADQLEMVTELLGGDMVNQSFICDPDDETFIKNIIIQDCMWSGESAAAKLVSEKLASYQAARKDSGSPNPARGHSVCSTSSLYLQDLSAAASECIDPSVVFPYPLNDSSSPKSCASQDSSAFSPSSDSLLSSTESSPQGSPEPLVLHEETPPTTSSDSEEEQEDEEEIDVVSVEKRQAPGKRSESGSPSAGGHSKPPHSPLVLKRCHVSTHQHNYAAPPSTRKDYPAAKRVKLDSVRVLRQISNNRKCTSPRSSDTEENVKRRTHNVLERQRRNELKRSFFALRDQIPELENNEKAPKVVILKKATAYILSVQAEEQKLISEEDLLRKRR EQLKHKLEQLR.

In some embodiments, the MYC polypeptide sequence comprises a MYCpolypeptide sequence from a non-human species. In some embodiments, thenon-human species is selected from the group consisting of ape, monkey,mouse, rat, hamster, guinea pig, rabbit, cat, dog, pig, sheep, goat,cow, and horse species. In some embodiments, the MYC polypeptidesequence comprises the sequence shown below, which is from Chlorocebussabaeus (green monkey) (XP_007999715.1):

MPLNVSFTNRNYDLDYDSVQPYFYCDEEENFYQQQQQSELQPPAPSEDIWKKFELLPTPPLSPSRRSGLCSPSYVAVTPFSPRGDNDGGGGSFSTADQLEMVTELLGGDMVNQSFICDPDDETFIKNIIIQDCMWSGESAAAKLVSEKLASYQAARKDSGSPNPARGHSVCSTSSLYLQDLSAAASECIDPSVVFPYPLNDSSSPKSCASPDSSAFSPSSDSLLSSTESSPQASPEPLVLHEETPPTTSSDSEEEQEDEEEIDVVSVEKRQAPGKRSESGSPSAGGHSKPPHSPLVLKRCHVSTHQHNYAAPPSTRKDYPAAKRVKLDSVRVLRQISNNRKCTSPRSSDTEENVKRRTHNVLERQRRNELKRSFFALRDQIPELENNEKAPKVVILKKATAYILSVQAEEQKLISEKDLLRKR REQLKHKLEQLRNSCA

In some embodiments, the MYC polypeptide sequence comprises the sequenceshown below:

(SEQ ID NO: 9) PLNVSFTNRNYDLDYDSVQPYFYCDEEENFYQQQQQSELQPPAPSEDIWKKFELLPTPPLSPSRRSGLCSPSYVAVTPFSLRGDNDGGGGSFSTADQLEMVTELLGGDMVNQSFICDPDDETFIKNIIIQDCMWSGFSAAAKLVSEKLASYQAARKDSGSPNPARGHSVCSTSSLYLQDLSAAASECIDPSVVFPYPLNDSSSPKSCASQDSSAFSPSSDSLLSSTESSPQASPEPLVLHEETPPTTSSDSEEEQEDEEEIDVVSVEKRQAPGKRSESGSPSAGGHSKPPHSPLVLKRCHVSTHQHNYAAPPSTRKDYPAAKRVKLDSVRVLRQISNNRKCTSPRSSDTEENVKRRTHNVLERQRRNELKRSFFALRDQIPELENNEKAPKVVILKKATAYILSVQAEEQKLISEKDLLRKRR EQLKHKLEQLR.

In some embodiments, a MYC polypeptide comprises an amino acid sequencethat is at least 40% to 100% identical, e.g., at least 40%, 45%, 50%,55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,90%, 91%, 92%, 94%, 95%, 96%, 97%, 98%, 99%, or any other percent fromabout 40% to about 100% identical to the sequence of NCBI AccessionNumber NP002458.2 (SEQ ID NO: 2). In some embodiments, a MYC polypeptiderefers to a polymer of 420, 421, 422, 423, 424, 425, 426, 427, 428, 429,430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443,444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454 consecutive aminoacids of NP002458.2 (SEQ ID NO: 2). In some embodiments, a MYCpolypeptide refers to a polymer of 435 amino acids of NP002458.2 (SEQ IDNO: 2) that has not undergone any post-translational modifications. Insome embodiments, a MYC polypeptide refers to a polymer of 435 aminoacids of NP002458.2 (SEQ ID NO: 2) that has undergone post-translationalmodifications. In some embodiments, the MYC polypeptide is 48,804 kDa.In some embodiments, the MYC polypeptide contains a basicHelix-Loop-Helix Leucine Zipper (bHLH/LZ) domain. In some embodiments,the bHLH/LZ domain comprises the sequence ofELKRSFFALRDQIPELENNEKAPKVVILKKATAYILSVQAEEQKLISEEDLLRKRREQLKH KLEQLR(SEQ ID NO: 5). In some embodiments, the MYC polypeptide is atranscription factor (e.g., Transcription Factor 64). In someembodiments, the MYC polypeptide contains a E-box DNA binding domain. Insome embodiments, the MYC polypeptide binds to a sequence comprisingCACGTG. In some embodiments, the MYC polypeptide promotes one or more ofcell survival and/or proliferation. In some embodiments, a MYCpolypeptide includes one or more of those described above, and includesone or more post-translational modifications (e.g., acetylation). Insome embodiments, the MYC polypeptides comprise one or more additionalamino acid residues at the N-terminus or C-terminus of the polypeptide.In some embodiments, the MYC polypeptides are fusion proteins. In someembodiments, the MYC polypeptides are linked to one or more additionalpeptides at the N-terminus or C-terminus of the polypeptide.

Proteins suitable for use in the methods described herein also includesfunctional variants, including proteins having between 1 to 15 aminoacid changes, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15amino acid substitutions, deletions, or additions, compared to the aminoacid sequence of any protein described herein. In other embodiments, thealtered amino acid sequence is at least 75% identical, e.g., 75%, 76%,77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to theamino acid sequence of any protein inhibitor described herein. Suchsequence-variant proteins are suitable for the methods described hereinas long as the altered amino acid sequence retains sufficient biologicalactivity to be functional in the compositions and methods describedherein. Where amino acid substitutions are made, the substitutions maybe conservative amino acid substitutions. Among the common, naturallyoccurring amino acids, for example, a “conservative amino acidsubstitution” is illustrated by a substitution among amino acids withineach of the following groups: (1) glycine, alanine, valine, leucine, andisoleucine, (2) phenylalanine, tyrosine, and tryptophan, (3) serine andthreonine, (4) aspartate and glutamate, (5) glutamine and asparagine,and (6) lysine, arginine and histidine. The BLOSUM62 table is an aminoacid substitution matrix derived from about 2,000 local multiplealignments of protein sequence segments, representing highly conservedregions of more than 500 groups of related proteins (Henikoff et al.,(1992), Proc. Natl Acad. Sci. USA, 89:10915-10919). Accordingly, theBLOSUM62 substitution frequencies are used to define conservative aminoacid substitutions that, in some embodiments, are introduced into theamino acid sequences described or disclosed herein. Although it ispossible to design amino acid substitutions based solely upon chemicalproperties (as discussed above), the language “conservative amino acidsubstitution” preferably refers to a substitution represented by aBLOSUM62 value of greater than −1. For example, an amino acidsubstitution is conservative if the substitution is characterized by aBLOSUM62 value of 0, 1, 2, or 3. According to this system, preferredconservative amino acid substitutions are characterized by a BLOSUM62value of at least 1 (e.g., 1, 2 or 3), while more preferred conservativeamino acid substitutions are characterized by a BLOSUM62 value of atleast 2 (e.g., 2 or 3).

The phrases “E-box sequence” and “enhancer box sequence” are usedinterchangeably herein and mean the nucleotide sequence CANNTG, whereinN is any nucleotide. In certain instances, the E-box sequence comprisesCACGTG. In certain instances, the basic helix-loop-helix domain of atranscription factor encoded by MYC binds to the E-box sequence. Incertain instances the E-box sequence is located upstream of a gene(e.g., p21, Bc1-2, or ornithine decarboxylase). In certain instances,the MYC polypeptide contains an E-box DNA binding domain. In certaininstances, the E-box DNA binding domain comprises the sequence ofKRRTHNVLERQRRN (SEQ ID NO: 6). In certain instances, the binding of thetranscription factor encoded by MYC to the E-box sequence, allows RNApolymerase to transcribe the gene downstream of the E-box sequence.

The term “MYC activity” or “MYC biological activity” or “biologicallyactive MYC” includes one or more of enhancing or inducing cell survival,cell proliferation, and/or antibody production. By way of example andnot by way of limitation, MYC activity includes enhancement of expansionof anti-CD3 and anti-CD28 activated T-cells and/or increasedproliferation of long-term self-renewing hematopoietic stem cells. MYCactivity also includes entry into the nucleus of a cell, binding to anucleic acid sequence (e.g., binding an E-box sequence), and/or inducingexpression of MYC target genes.

The terms “patient,” “subject,” “individual,” and the like are usedinterchangeably herein, and refer to an animal, typically a mammal. In apreferred embodiment, the patient, subject, or individual is a mammal.In a particularly preferred embodiment, the patient, subject orindividual is a human.

The terms “protein transduction domain (PTD)” or “transporter peptidesequence” (also known as cell permeable proteins (CPP) or membranetranslocating sequences (MTS)) are used interchangeably herein to referto small peptides that are able to ferry much larger molecules intocells independent of classical endocytosis. In some embodiments, anuclear localization signal can be found within the protein transductiondomain, which mediates further translocation of the molecules into thecell nucleus.

The terms “treating” or “treatment” as used herein covers the treatmentof a disease in a subject, such as a human, and includes: (i) inhibitinga disease, i.e., arresting its development; (ii) relieving a disease,i.e., causing regression of the disease; (iii) slowing progression ofthe disease; and/or (iv) inhibiting, relieving, or slowing progressionof one or more symptoms of the disease.

It is also to be appreciated that the various modes of treatment orprevention of medical diseases and conditions as described are intendedto mean “substantial,” which includes total but also less than totaltreatment or prevention, and wherein some biologically or medicallyrelevant result is achieved. The treatment may be a continuous prolongedtreatment for a chronic disease or a single, or few time administrationsfor the treatment of an acute condition.

The term “therapeutic” as used herein means a treatment and/orprophylaxis. A therapeutic effect is obtained by suppression, remission,or eradication of a disease state.

II. Compositions Comprising Nanoparticulate MYC Peptides

Disclosed herein are compositions comprising MYC-containing polypeptidesformulated as biologically active, stable nanoparticles, and methods ofmaking and using the compositions. In some embodiments, theMYC-containing polypeptide is a fusion of a MYC polypeptide and aprotein transduction domain, e.g., HIV TAT. In some embodiments, the MYCfusion polypeptide also includes one or more tag sequences. In someembodiments, the MYC fusion polypeptide comprises SEQ ID NO: 1. In someembodiments, the MYC fusion polypeptide comprises SEQ ID NO: 10.

As discussed in more detail below and as illustrated in the Examples,biologically active MYC-containing polypeptide compositions of thepresent technology include, in some embodiments, nanoparticles of about90-140 nm with molecular mass of about 10⁴-10⁶ daltons. In someembodiments, the particles include about 200 molecules of MYC-containingpolypeptide.

In some embodiments, the biologically active nanoparticulatecompositions include post-translationally modified MYC protein. By wayof example, but not by way of limitation, in some embodiments, theprotein includes at least one acetyl group.

A. Expression and Purification of MYC-Containing Polypeptides

Provided herein are methods for the production of MYC-containingpolypeptides for use in nanoparticle compositions provided. In themethods of the invention, MYC-containing polypeptide is recombinantlyproduced by microbial fermentation. In some embodiments microbialfermentation is performed in a fermentation volume of from about 1 toabout 10,000 liters, for example, a fermentation volume of about 10 toabout 1000 liters. The fermentation can utilize any suitable microbialhost cell and culture medium. In exemplary embodiments, E. coli isutilized as the microbial host cell. In alternative embodiments, othermicroorganisms can be used, e.g., S. cerevisiae, P. pastoris,Lactobacilli, Bacilli and Aspergilli. In an exemplary embodiment themicrobial host cell is BL-21 Star™ E. coli strain (Invitrogen). In anexemplary embodiment the microbial host cell is BLR DE3 E. coli. strain

In some embodiments the host cells are modified to provide tRNAs forrare codons, which are employed to overcome host microbial cell codonbias to improve translation of the expressed proteins. In exemplaryembodiments, the host cells (e.g., E. coli) transformed with a plasmid,such as pRARE (CamR), which express tRNAs for AGG, AGA, AUA, CUA, CCC,GGA codons. Additional, suitable plasmids or constructs for providingtRNAs for particular codons are known in the art and can be employed inthe methods provided.

Integrative or self-replicative vectors may be used for the purpose ofintroducing the MYC-containing polypeptide expression cassette into ahost cell of choice. In an expression cassette, the coding sequence forthe MYC-containing polypeptide is operably linked to promoter, such asan inducible promoter. Inducible promoters are promoters that initiateincreased levels of transcription from DNA under their control inresponse to some change in culture conditions, e.g., the presence orabsence of a nutrient or a change in temperature. In some embodiments,the nucleic acid encoding the MYC-containing polypeptide is codonoptimized for bacterial expression.

Exemplary promoters that are recognized by a variety of potential hostcells are well known. These promoters can be operably linked toMYC-containing polypeptide-encoding DNA by removing the promoter fromthe source DNA, if present, by restriction enzyme digestion andinserting the isolated promoter sequence into the vector. Promoterssuitable for use with microbial hosts include, but are not limited to,the β-lactamase and lactose promoter systems (Chang et al., (1978)Nature, 275:617-624; Goeddel et al., (1979) Nature, 281: 544), alkalinephosphatase, a tryptophan (trp) promoter system (Goeddel (1980) NucleicAcids Res. 8: 4057; EP 36,776), and hybrid promoters such as the tacpromoter (deBoer et al., (1983) Proc. Natl. Acad. Sci. USA 80: 21-25).Any promoter for suitable for expression by the selected host cell canbe used. Nucleotide sequences for suitable are published, therebyenabling a skilled worker operably to ligate them to DNA encodingMYC-containing polypeptide (see, e.g., Siebenlist et al., (1980) Cell20: 269) using linkers or adaptors to supply any required restrictionsites. In exemplary embodiments, promoters for use in bacterial systemscan contain a Shine-Dalgarno (S.D.) sequence operably linked to thecoding sequence. In some embodiments, the inducible promoter is the lacZpromoter, which is induced with Isopropyl β-D-1-thiogalactopyranoside(IPTG), as is well-known in the art. Promoters and expression cassettescan also be synthesized de novo using well known techniques forsynthesizing DNA sequences of interest. In an exemplary embodiment, theexpression vector for expression of the MYC-containing polypeptidesherein is pET101/D-Topo (Invitrogen).

For expression of the MYC-containing polypeptides, the microbial hostcontaining the expression vector encoding the MYC-containing polypeptideis typically grown to high density in a fermentation reactor. In someembodiments, the reactor has controlled feeds for glucose. In someembodiments, a fermenter inoculum is first cultured in mediumsupplemented with antibiotics (e.g., overnight culture). The fermenterinoculum is then used to inoculate the fermenter culture for expressionof the protein. At an OD600 of at least about 15, usually at least about20, at least 25, at least about 30 or higher, of the fermenter culture,expression of the recombinant protein is induced. In exemplaryembodiments, where the inducible promoter is the lacZ promoter, IPTG isadded to the fermentation medium to induce expression of theMYC-containing polypeptide. Generally, the IPTG is added to thefermenter culture at an OD600 which represents logarithmic growth phase.

In certain embodiments of the methods provided, induced proteinexpression is maintained for around about 2 to around about 5 hours postinduction, and can be from around about 2 to around about 3 hourspost-induction. Longer periods of induction may be undesirable due todegradation of the recombinant protein. The temperature of the reactionmixture during induction is preferably from about 28° C. to about 37°C., usually from about 30° C. to about 37° C. In particular embodiments,induction is at about 37° C.

The MYC-containing polypeptide is typically expressed as cytosolicinclusion bodies in microbial cells. To harvest inclusion bodies, a cellpellet is collected by centrifugation of the fermentation culturefollowing induction, frozen at −70° C. or below, thawed and resuspendedin disruption buffer. The cells are lysed by conventional methods, e.g.,sonication, homogenization, etc. The lysate is then resuspended insolubilization buffer, usually in the presence of urea at aconcentration effective to solubilize proteins, e.g., from around about5M, 6M, 7M, 8M, 9M or greater. Resuspension may require mechanicallybreaking apart the pellet and stirring to achieve homogeneity. In someembodiments, the cell pellet is directly resuspended in urea buffer andmixed until homogenous. In some embodiments, theresuspension/solubilization buffer is 8M Urea, 50 mM Phosphate pH 7.5and the suspension is passed through a homogenizer.

In some embodiments, the homogenized suspension is sulfonylated. Forexample, in some embodiments, the homogenized suspension is adjusted toinclude 200 mM Sodium Sulfite and 10 mM Sodium Tetrathionate. Thesolution is then mixed at room temperature until homogeneous. The mixedlysate is then mixed for an additional period of time to complete thesulfonylation (e.g., at 2-8° C. for ≥12 hours). The sulfonylated lysatewas then centrifuged for an hour. The supernatant containing thesulfonylated MYC-containing polypeptides is then collected bycentrifugation and the cell pellet discarded. The supernatant is thenpassed through a filter, e.g., 0.22 μm membrane filter to clarify thelysate.

The solubilized protein is then purified. Purification methods mayinclude affinity chromatography, reverse phase chromatography, gelexclusion chromatography, and the like. In some embodiments, affinitychromatography is used. For example, the protein is provided with anepitope tag or histidine 6 tag for convenient purification. In thepresent methods, exemplary myc-containing polypeptide comprise histidine6 tag for purification using Ni affinity chromatography using Ni-resin.

In exemplary embodiments, the Ni-resin column is equilibrated in abuffer containing urea. In some embodiments, the equilibration buffer is6M Urea, 50 mM Phosphate, 500 mM NaCl, and 10% Glycerol solution. Thesulfonylated and clarified supernatant comprising the MYC-containingpolypeptide is then loaded onto the Ni-resin column. The column is thenwashed with a wash buffer, e.g., 6M Urea, 50 mM Phosphate, 10% Glycerol,500 mM NaCl, pH 7.5. The column was then washed with sequential washbuffers with decreasing salt concentration. For example, exemplarysubsequent washed can include 6M Urea, 50 mM Phosphate, 10% Glycerol,and 2M NaCl, pH 7.5, followed another wash of 6M Urea, 50 mM Phosphate,10% Glycerol, 50 mM NaCl, and 30mM Imidazole, pH 7.5.

Following sequential application of the wash buffers the MYC-containingpolypeptide is eluted from the column by addition of elution buffer,e.g., 6M Urea, 50 mM Phosphate, 10% Glycerol, and 50 mM NaCl, pH 7.5with a gradient from 100 to 300 mM Imidazole, and collecting fractions.The protein containing fractions to be pooled are then filtered througha 0.22 μm membrane. Assessment of protein yield can be measured usingany suitable method, e.g., spectrophotometry at UV wavelength 280.

In some embodiments, one or more additional purification methods can beemployed to further purify the isolated MYC-containing polypeptides. Inexemplary embodiments, the pooled fractions from the Ni-Sepharosechromatography step are further purified by anion exchangechromatography using a Q-Sepharose resin. In some embodiments, the poolis prepared for loading onto the Q-Sepharose column by diluting thesamples to the conductivity of the Q sepharose buffer (17.52+/−1 mS/cm)with the second wash buffer (e.g., 6M Urea, 50 mM Phosphate, 10%Glycerol, 2M NaCl, pH 7.5) from the Ni Sepharose chromatography step.The diluted pool is then loaded onto the Q-Sepharose column, followed bytwo chase steps using a chase buffer (e.g., 6M Urea, 50 mM Phosphate,300 mM NaCl, and 10% Glycerol), with further sequential applications ofthe chase buffer until the UV trace reaches baseline, indicating thatthe protein has eluted from the column.

B. Refolding of MYC-Containing Polypeptides into Nanoparticles

Compositions of the present technology can be prepared from isolatedMYC-containing polypeptide according to the following methods.

In some embodiments, the MYC-containing polypeptides are refolded toproduce a biologically active nanoparticle. In some embodiments, themethod comprises Tangential Flow Filtration (TFF), or Cross FlowFiltration. TFF is a process which uses a pump to circulate a sampleacross the surface of membranes (i.e. “tangential” to the membranesurface) housed in a multilevel structure (cassette). The appliedtransmembrane pressure acts as the driving force to transport solute andsmall molecules through the membrane. The cross flow of liquid over themembrane surface sweeps retaining molecules from the surface, keepingthem in the circulation stream.

In some embodiments, the MYC-containing polypeptides are denatured witha denaturing agent, such as urea. The MYC-containing polypeptides arethen refolded using multiple TFF steps across aultrafiltration/diafiltration (UFDF) membrane and sequential additionsof refold buffers having decreasing concentrations of the denaturingagent, e.g., urea. In some embodiments, the urea concentration of thesequential refold buffers decreases from about 3M Urea to <0.001M or nourea. In some embodiments, the sequential refold buffers comprisePhosphate, NaCl, Glycerol, GSH, (Reduced Glutathione) and GSSG (OxidizedGlutathione)). In some embodiments, the refold buffers comprise about 50mM Phosphate. In some embodiments, the refold buffers comprise an alkaliearth metal salt. In some embodiments, alkali earth metal salt is a saltof sodium (Na), lithium (Li) or potassium (K). In some embodiments, therefold buffers comprise a sodium salt, such as NaCl. In someembodiments, the refold buffers comprise between about 100 mM to 2Mconcentration of an alkali earth metal salt. In some embodiments, therefold buffers comprise between about 200 mM to 1M concentration of analkali earth metal salt. In some embodiments, the refold bufferscomprise between about 500 mM to 1M concentration of an alkali earthmetal salt. In some embodiments, the refold buffers comprise betweenabout 100 mM to 2M concentration of NaCl. In some embodiments, therefold buffers comprise between about 200 mM to 1M concentration ofNaCl. In some embodiments, the refold buffers comprise between about 200mM to 800 mM concentration of NaCl. In particular embodiments, therefold buffers comprise about 200-500 mM NaCl. In some embodiments, therefold buffers comprise about 500 mM NaCl. In some embodiments, therefold buffers have an osmolarity between about 300 mOsm and 1000 mOsm.In some embodiments, the refold buffers comprise about 1 to 20%Glycerol. In some embodiments, the refold buffers comprise about 10%Glycerol. In some embodiments, the refold buffers comprise about 0.1 to50 mM GSH, (Reduced Glutathione). In some embodiments, the refoldbuffers comprise about 5 mM GSH, (Reduced Glutathione). In someembodiments, the refold buffers comprise about 0.1 to 50 mM GSSG(Oxidized Glutathione)). In some embodiments, the refold bufferscomprise about 1 mM GSSG (Oxidized Glutathione)). In exemplaryembodiments, the refold buffers comprise 50 mM Phosphate, 500 mM NaCl,10% Glycerol, 5 mM GSH, (Reduced Glutathione), and 1 mM GSSG (OxidizedGlutathione)). In some embodiments, the refold buffers have a pH valueof between about 5.0 and 8.0. In some embodiments, the refold buffershave a pH value of 7.5.

In an exemplary embodiment, the MYC-containing polypeptides are refoldedusing three TTF steps across a ultrafiltration/diafiltration (UFDF)membrane. In an exemplary embodiment, the first refold step involves anexchange of a refold buffer 1 (e.g., 3M Urea, 50 mM Phosphate, 500 mMNaCl, 10% Glycerol, 5 mM GSH, (Reduced Glutathione) 1 mM GSSG (OxidizedGlutathione)) over the course of about 120 minutes. In some embodiments,the second refold step involves the exchange of refold buffer 2 (1.5MUrea, 50 mM Phosphate, 500 mM NaCl, 10% Glycerol, 5 mM GSH, (ReducedGlutathione) 1 mM GSSG (Oxidized Glutathione)) over the course ofapproximately 120 minutes, followed by ˜120 minutes of recirculation. Insome embodiments, the third refold step involves the exchange of refoldbuffer 3 (50 mM Phosphate, 500 mM NaCl, 10% Glycerol, 5 mM GSH, (ReducedGlutathione) 1 mM GSSG (Oxidized Glutathione)) over the course ofapproximately 120 minutes, followed by 12 hours of recirculation.

After the final refolding step is complete, the final refold solutioncan be filtered through a 0.2 μm membrane and protein concentrationadjusted.

Following refolding, the nanoparticle formulation contains nanoparticleshaving a wide range of sizes and stabilities. Thus, in some embodiments,an incubation step, or equilibration step, is performed after the thirdrefold step. For example, in some embodiments, the refoldedMYC-containing polypeptides are maintained in the third refold bufferfor a period of time sufficient to produce biologically activenanoparticles having a number average diameter of between about 80 nmand about 150 nm. Without being bound by theory, it is believed that theequilibration step allows nanoparticle formulation to equilibrate intostable nanoparticles having narrower size range and more stability. Insome embodiments, the number average diameter of these stablenanoparticles is between about 80 nm and about 150 nm. In someembodiments, the equilibration step is performed for a length of time ofat least 5, 6, 7, 8, 9, 10, 11, or 12 hours or more. In exemplaryembodiments, the equilibration is step is performed for at least orabout 10-12 hours. In exemplary embodiments, the equilibration is stepinvolves gently stirring the nanoparticle formulation of refoldedMYC-containing polypeptides in the third refold buffer. In exemplaryembodiments, the equilibration is step involves stirring the formulationthe MYC-containing polypeptides in the third refold buffer at less than1000 rpm.

In some embodiments, an additional exchange of final refold buffer 3 isperformed to exchange the refold buffer with a formulation buffersuitable for administration, such as a buffer suitable for injection. Inexemplary embodiments, refolded TAT-MYC in refold buffer 3 is dialyzedagainst a suitable formulation buffer. In exemplary embodiments, therefolded TAT-MYC in refold buffer 3 is dialyzed using TFF across aultrafiltration/diafiltration (UFDF) membrane. In exemplary embodiments,the formulation buffer comprises a buffering agent. In exemplaryembodiments, the buffering agent is selected from among sodiumphosphate, potassium phosphate, histidine, and citrate.

In exemplary embodiments, refolded TAT-MYC is stable in a formulationhaving a pH value of between 5.5 and 8.0. In exemplary embodiments,refolded TAT-MYC is stable in a formulation having a pH value of between6.0 and 8.0. In exemplary embodiments, refolded TAT-MYC is stable in aformulation having a pH value of about 6.0, about 6.5, about 7.0, about7.5, or about 8.0. In exemplary embodiments, refolded TAT-MYC is stablein a formulation having a pH value of about pH 7.5. In exemplaryembodiments, refolded TAT-MYC is stable in a formulation of about pH7.5+/−pH 0.3.

In exemplary embodiments, the refolded TAT-MYC is stable in aformulation having an osmolality greater than 300 mOsm. In exemplaryembodiments, the refolded TAT-MYC is stable in a formulation having anosmolality greater than 400 mOsm. In exemplary embodiments, the refoldedTAT-MYC is stable in a formulation having an osmolality between 300 mOsmand 1000 mOsm, such as between 400 mOsm and 800 mOsm.

In exemplary embodiments, the refolded TAT-MYC is stable in aformulation comprising greater than 100 mM NaCl. In exemplaryembodiments, the refolded TAT-MYC is stable in a formulation comprisinggreater than 150 mM NaCl. In exemplary embodiments, the NaClconcentration of a refolded TAT-MYC formulation is about 150 mM, 200 mM,250 mM, 300 mM, 350 mM, 400 mM, 450 mM, 500 mM or 550 mM NaCl. Inexemplary embodiments, the NaCl concentration of a refolded TAT-MYCformulation is about 500 mM+/−50 mM NaCl.

In some embodiments, refolded TAT-MYC can be stored up to aconcentration of about 1.2 mg/mL. As used herein, “stability” withreference to a storage condition refers the ability of the refoldedTAT-MYC to retain at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% ormore of its MYC biological activity following storage, compared to theMYC biological activity of the refolded TAT-MYC prior to storage. Insome embodiments, refolded TAT-MYC is stable at −80° C. for up to 2years. In some embodiments, once thawed, refolded TAT-MYC is stable whenstored at 4° C. for up to 1 month. In exemplary embodiments, oncethawed, refolded TAT-MYC is stable when stored at 4° C. for up to 2months. In exemplary embodiments, once thawed, refolded TAT-MYC isstable when stored at 4° C. for up to 3 months. In exemplaryembodiments, once thawed, refolded TAT-MYC is stable when stored at 4°C. for up to 4 months. In exemplary embodiments, once thawed, refoldedTAT-MYC is stable when stored at 4° C. for up to 5 months. In exemplaryembodiments, once thawed, refolded TAT-MYC is stable when stored at 4°C. for up to 6 months or longer. In exemplary embodiments, once thawed,refolded TAT-MYC is stable when stored at 4° C. for up to 100 days orlonger. Accordingly, the stability of the refolded TAT-MYC providedherein is significantly increased compared to the stability of awild-type MYC polypeptide, which has a half-life of approximately 34minutes (Kaptein et al. (1996) JBC 271, 18875-18884).

C. Nanoparticle Size

The size and mass of the nanoparticles of the present technology can bedetermined by methods well known in the art. By way of example, but notby way of limitation, such methods include size exclusionchromatography, high performance liquid chromatography, dynamic lightscattering, nanoparticle tracking analysis, and electron microscopy.

In some embodiments, the average particle size of the nanoparticles inthe formulation is between about 70 nm and about 140 nm, such as between70 nm and 140 nm. In some embodiments, the average particle size of thenanoparticles in the formulation is between about 80 nm and about 120nm, such as between 80 nm and 120 nm. In some embodiments, the averageparticle size of the nanoparticles in the formulation is between about80 nm and about 110 nm, such as between 80 nm and 110 nm. In someembodiments, the average particle size of the nanoparticles in theformulation is between about 80 nm and about 90 nm, such as between 80nm and 90 nm. In some embodiments, the average particle size of thenanoparticles in the formulation is about 84 nm or 84 nm. In someembodiments, the average particle size of the nanoparticles in theformulation is between about 100 nm and about 120 nm, such as between100 nm and 120 nm. In some embodiments, the average particle size of thenanoparticles in the formulation is about 111 nm or 111 nm.

In some embodiments, the average molecular weight of the particles inthe formulation is between about 10³-10⁷ daltons, such as between10³-10⁷ daltons. In some embodiments, the average molecular weight ofthe particles in the formulation is between about 10⁴-10⁷ daltons, suchas between 10⁴-10⁷ daltons. In some embodiments, the average molecularweight of the particles in the formulation is between about 10⁵-10⁷daltons, such as between 10⁵-10⁷ daltons. In some embodiments, theaverage molecular weight of the particles in the formulation is betweenabout 10⁶-10⁷ daltons, such as between 10⁶-10⁷ daltons. In someembodiments, the average molecular weight of the particles in theformulation is about 2×10⁶ or 2×10⁶ daltons.

In some embodiments, less than about 0.01% or less than 0.01% of thenanoparticles within the composition have a particle size greater than200 nm, greater than 300 nm, greater than 400 nm, greater than 500 nm,greater than 600 nm, greater than 700 nm, or greater than 800 nm. Insome embodiments, less than about 0.001% of the nanoparticles within thecomposition have a particle size greater than 200 nm, greater than 300nm, greater than 400 nm, greater than 500 nm, greater than 600 nm,greater than 700 nm, or greater than 800 nm. In some embodiments, lessthan about 0.01% or less than 0.01% of the nanoparticles within thecomposition have a particle size greater than 800 nm. In someembodiments, less than about 0.001% or less than 0.001% of thenanoparticles within the composition have a particle size greater than800 nm.

In some embodiments, less than about 0.01% or less than 0.01% of thenanoparticles within a formulation have a particle size less than 80 nm,less than 70 nm, less than 60 nm, less than 50 nm, less than 40 nm, lessthan 30 nm, less than 20 nm, or less than 10 nm. In some embodiments,less than about 0.001% or less than 0.001% of the nanoparticles withinthe composition have a particle size less than 80 nm, less than 70 nm,less than 60 nm, less than 50 nm, less than 40 nm, less than 30 nm, lessthan 20 nm, or less than 10 nm. In some embodiments, less than about0.01% or less than 0.01% of the nanoparticles within the compositionhave a particle size less than 50 nm. In some embodiments, less thanabout 0.001% or less than 0.001% of the nanoparticles within thecomposition have a particle size less than 50 nm.

D. MYC Fusion Proteins

In some embodiments, the biologically active nanoparticulatecompositions of MYC-containing polypeptide comprise a MYC fusionprotein. In some embodiments, the MYC fusion protein comprises a proteintransduction domain, a MYC polypeptide that promotes one or more of cellsurvival or proliferation, and optionally a protein tag domain, e.g.,one or more amino acid sequences that facilitate purification of thefusion protein. In some embodiments, a cell contacted with MYCpolypeptide (e.g., a nanoparticulate formulation of the presenttechnology) exhibits increased survival time (e.g., as compared to anidentical or similar cell of the same type that was not contacted withMYC), and/or increased proliferation (e.g., as compared to an identicalor similar cell of the same type that was not contacted with MYC).

In some embodiments, the fusion protein comprises (a) a proteintransduction domain; and (b) a MYC polypeptide sequence. In someembodiments, the fusion peptide is a peptide of Formula (I):

protein transduction domain-MYC polypeptide sequence.

In some embodiments, a fusion peptide disclosed herein comprises (a) aprotein transduction domain; (b) a MYC polypeptide sequence; and (c) oneor more molecules that link the protein transduction domain and the MYCpolypeptide sequence. In some embodiments, the fusion peptide is apeptide of Formula (II):

protein transduction domain-X-MYC polypeptide sequence,

wherein -X- is molecule that links the protein transduction domain andthe MYC polypeptide sequence. In some embodiments, -X- is at least oneamino acid.

In some embodiments, a fusion peptide disclosed herein comprises (a) aprotein transduction domain; (b) a MYC polypeptide sequence; (c) atleast two protein tags; and (d) optionally linker(s). In someembodiments, the fusion peptide is a peptide of Formula (III-VI):

protein transduction domain-X-MYC polypeptide sequence-X-protein tag1-X-protein tag 2 (Formula (III)), or

protein transduction domain-MYC polypeptide sequence-X-protein tag1-X-protein tag 2 (Formula (IV)), or

protein transduction domain-MYC polypeptide sequence-protein tag1-X-protein tag 2 (Formula (V)), or

protein transduction domain-MYC polypeptide sequence-protein tag1-protein tag 2 (Formula (VI)),

wherein -X- is a linker. In some embodiments, -X- is one or more aminoacids.

In some embodiments, a fusion peptide disclosed herein comprises (a) aprotein transduction domain; (b) a MYC polypeptide sequence; (c) a6-histidine tag; (d) a V5 epitope tag: and (e) optionally linker(s). Insome embodiments, the fusion peptide is a peptide of Formula (VII-XIV):

protein transduction domain-X-MYC polypeptide sequence-X-6-histidinetag-X-V5 epitope tag (Formula (VII)), or

protein transduction domain-MYC polypeptide sequence-X-6-histidinetag-X-V5 epitope tag (Formula (VIII)), or

protein transduction domain-MYC polypeptide sequence-6-histidinetag-X-V5 epitope tag (Formula (IX)), or

protein transduction domain-MYC polypeptide sequence-6-histidine tag-V5epitope tag (Formula (X)),

protein transduction domain-X-MYC polypeptide sequence-X-V5 epitopetag-X-6-histidine tag (Formula (XI)), or

protein transduction domain-MYC polypeptide sequence-X-V5 epitopetag-X-6-histidine tag (Formula (XII)), or

protein transduction domain-MYC polypeptide sequence-V5 epitopetag-X-6-histidine tag (Formula (XIII)), or

protein transduction domain-MYC polypeptide sequence-V5 epitopetag-6-histidine tag (Formula (XIV)),

wherein -X- is a linker. In some embodiments, -X- is one or more aminoacids.

As noted above, in some embodiments, the MYC fusion protein comprisesone or more linker sequences. The linker sequences can be employed tolink the protein transduction domain, MYC polypeptide sequence, V5epitope tag and/or 6-histidine tag of the fusion protein. In someembodiments, the linker comprises one or more amino acids. In someembodiments, the amino acid sequence of the linker comprises KGELNSKLE(SEQ ID NO: 11). In some embodiments, the linker comprises the aminoacid sequence of RTG.

-   -   1. Protein Transduction Domain (PTD)

In some embodiments, the MYC fusion protein includes a proteintransduction domain. Peptide transport provides an alternative fordelivery of small molecules, proteins, or nucleic acids across the cellmembrane to an intracellular compartment of a cell. One non-limitingexample and well-characterized protein transduction domain (PTD) is aTAT-derived peptide. Frankel et al., (see, e.g., U.S. Pat. Nos.5,804,604, 5,747,641, 5,674,980, 5,670,617, and 5,652,122) demonstratedtransport of a cargo protein (β-galactosidase or horseradish peroxidase)into a cell by conjugating a peptide containing amino acids 48-57 of TATto the cargo protein. In some embodiments, TAT protein transductiondomain comprises an amino acid sequence of MRKKRRQRRR (SEQ ID NO: 7).

Another non-limiting example of a PTD is penetratin. Penetratin cantransport hydrophilic macromolecules across the cell membrane (Derossiet al., Trends Cell Biol., 8:84-87 (1998) incorporated herein byreference in its entirety). Penetratin is a 16 amino acid peptide thatcorresponds to amino acids 43-58 of the homeodomain of Antennapedia, aDrosophila transcription factor which is internalized by cells inculture.

Yet another non-limiting example of a PTD is VP22. VP22, a tegumentprotein from Herpes simplex virus type 1 (HSV-1), has the ability totransport proteins and nucleic acids across a cell membrane (Elliot etal., Cell 88:223-233, 1997, incorporated herein by reference in itsentirety). Residues 267-300 of VP22 are necessary but may not besufficient for transport. Because the region responsible for transportfunction has not been identified, the entire VP22 protein is commonlyused to transport cargo proteins and nucleic acids across the cellmembrane (Schwarze et al., Trends Pharmacol Sci, 21:45-48, 2000).

In some embodiments, the MYC fusion polypeptide includes a proteintransduction domain. By way of example, but not by way of limitation, insome embodiments, the protein transduction domain comprises the proteintransduction domain of one or more of TAT, penetratin, VP22, vpr, EPTD,R9, R15, VP16, and Antennapedia. In some embodiments, the proteintransduction domain comprises the protein transduction domain of one ormore of TAT, penetratin, VP22, vpr, and EPTD. In some embodiments, theprotein transduction domain comprises the protein transduction domain ofat least one of TAT, penetratin, VP22, vpr, EPTD, R9, R15, VP16, andAntennapedia. In some embodiments, the protein transduction domaincomprises a synthetic protein transduction domain (e.g., polyarginine orPTD-5). In particular embodiments, the protein transduction domaincomprises a TAT protein transduction domain. In some embodiments, theprotein transduction domain is covalently linked to the MYC polypeptide.In some embodiments, the protein transduction domain is linked to theMYC polypeptide via a peptide bond. In some embodiments, the proteintransduction domain is linked to the MYC polypeptide via a linkersequence. In some embodiments, the linker comprises a short amino acidsequence. By way of example, but not by way of limitation, in someembodiments, the linker sequences is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10amino acids in length.

The MYC fusion protein of the present technology can be arranged in anydesired order. For example, in some embodiments, the MYC fusion proteincan be arranged in order of a) the protein transduction domain linked inframe to the MYC polypeptide, b) the MYC polypeptide linked in frame tothe V5 domain, and c) the V5 domain linked in frame to the 6-histidineepitope tag. In some embodiments, the MYC fusion protein has an order ofcomponents of a) the MYC polypeptide linked in frame to the proteintransduction domain, b) the protein transduction domain linked in frameto the V5 domain, and c) the V5 domain linked in frame to the6-histidine epitope tag. In some embodiments, additional amino acidsequences can be included between each of the sequences. In someembodiments, additional amino acids can be included at the start and/orend of the polypeptide sequences.

In some embodiments, the protein transduction domain is a TAT proteintransduction domain. In some embodiments, the protein transductiondomain is TAT_([48-57]). In some embodiments, the protein transductiondomain is TAT_([57-48]).

-   -   2. Protein Tag Domains

In some embodiments, the MYC fusion protein comprises a protein tagdomain that comprises one or more amino acid sequences that facilitatepurification of the fusion protein. In some embodiments, the protein tagdomain comprises one or more of a polyhistidine tag, and an epitope tag.By way of example, but not by way of limitation, exemplary tags includeone or more of a V5, a histidine-tag (e.g., a 6-histidine tag), HA(hemagglutinin) tags, FLAG tag, CBP (calmodulin binding peptide), CYD(covalent yet dissociable NorpD peptide), Strepll, or HPC (heavy chainof protein C). In some embodiments, the protein tag domain compriseabout 10 to 20 amino acids in length. In some embodiments, the proteintag domain comprises 2 to 40 amino acids in length, for example 6-20amino acids in length. In some embodiments, two of the above listed tags(for example, V5 and the his-tag) are used together to form the proteintag domain.

In some embodiments, the histidine tag is a 6-histidine tag. In someembodiments, the histidine tag comprises the sequence HHHHHH. In someembodiments, the fusion peptide disclosed herein comprises a V5 epitopetag. In some embodiments, the V5 tag comprises the amino acid sequenceof: GKPIPNPLLGLDST. In some embodiments, the V5 tag comprises the aminoacid sequence of IPNPLLGLD.

The protein tags may be added to the fusion protein disclosed herein byany suitable method. By way of example, but not by way of limitation, insome embodiments, a TAT-MYC polypeptide sequence is cloned into anexpression vector encoding one or more protein tags, e.g., a polyHis-tagand/or a V5 tag. In some embodiments, a polyhistidine tag and/or a V5tag is added by PCR (i.e., the PCR primers comprise a polyhistidinesequence and/ or V5 sequence).

C. Construction of MYC Fusion Peptides

MYC fusion peptides (e.g., TAT-MYC fusion peptide) disclosed herein maybe constructed by methods well known in the art. By way of example, butnot by way of limitation, a nucleotide sequence encoding a TAT-MYCfusion peptide may be generated by PCR. In some embodiments, a forwardprimer for a human MYC sequence comprises an in frame N-terminal9-amino-acid sequence of the TAT protein transduction domain (e.g.,RKKRRQRRR). In some embodiments, a reverse primer for a human MYCsequence is designed to remove the stop codon. In some embodiments, thePCR product is cloned into any suitable expression vector. In someembodiments, the expression vector comprises a polyhistidine tag and aV5 tag.

In some embodiments, a fusion peptide disclosed herein comprises (a)TAT, and (b) c-MYC. In some embodiments, a fusion peptide disclosedherein comprises (a) TAT_([48-57]), and (b) c-MYC. In some embodiments,a fusion peptide disclosed herein comprises (a) TAT_([57-48]), and (b)c-MYC.

In some embodiments, a fusion peptide disclosed herein comprises (a)TAT, (b) c-MYC, (c) linker(s), (d) V5 tag, and (e) 6-histidine tag. Insome embodiments, a fusion peptide disclosed herein comprises (a)TAT_([48-57]), (b) c-MYC, (c) linker(s), (d) V5 tag, and (e) 6-histidinetag. In some embodiments, a fusion peptide disclosed herein comprises(a) TAT_([57-48]), (b) c-MYC, (c) linker(s), (d) V5 tag, and (e)6-histidine tag.

In some embodiments, the MYC portion of the MYC fusion peptide comprisesany MYC polypeptide as described herein. In some embodiments, the MYCportion of the MYC fusion peptide comprises a MYC polypeptide sequencecomprising the sequence shown below:

(SEQ ID NO: 4) PLNVSFTNRNYDLDYDSVQPYFYCDEEENFYQQQQQSELQPPAPSEDIWKKFELLPTPPLSPSRRSGLCSPSYVAVTPFSLRGDNDGGGGSFSTADQLEMVTELLGGDMVNQSFICDPDDETFIKNIIIQDCMWSGESAAAKLVSEKLASYQAARKDSGSPNPARGHSVCSTSSLYLQDLSAAASECIDPSVVFPYPLNDSSSPKSCASQDSSAFSPSSDSLLSSTESSPQGSPEPLVLHEETPPTTSSDSEEEQEDEEEIDVVSVEKRQAPGKRSESGSPSAGGHSKPPHSPLVLKRCHVSTHQHNYAAPPSTRKDYPAAKRVKLDSVRVLRQISNNRKCTSPRSSDTEENVKRRTHNVLERQRRNELKRSFFALRDQIPELENNEKAPKVVILKKATAYILSVQAEEQKLISEEDLLRKRR EQLKHKLEQLR.

In some embodiments, the MYC fusion peptide comprises SEQ ID NO: 1. Insome embodiments, the MYC-fusion peptide is SEQ ID NO: 1.

(SEQ ID NO: 1) MRKKRRQRRRPLNVSFTNRNYDLDYDSVQPYFYCDEEENFYQQQQQSELQPPAPSEDIWKKFELLPTPPLSPSRRSGLCSPSYVAVTPFSLRGDNDGGGGSFSTADQLEMVTELLGGDMVNQSFICDPDDETFIKNIIIQDCMWSGFSAAAKLVSEKLASYQAARKDSGSPNPARGHSVCSTSSLYLQDLSAAASECIDPSVVFPYPLNDSSSPKSCASQDSSAFSPSSDSLLSSTESSPQGSPEPLVLHEETPPTTSSDSEEEQEDEEEIDVVSVEKRQAPGKRSESGSPSAGGHSKPPHSPLVLKRCHVSTHQHNYAAPPSTRKDYPAAKRVKLDSVRVLRQISNNRKCTSPRSSDTEENVKRRTHNVLERQRRNELKRSFFALRDQIPELENNEKAPKVVILKKATAYILSVQAEEQKLISEEDLLRKRREQLKHKLEQLRKGELNSKLEGKPIPNPLLGLDSTRTG HEIHHHH.

In some embodiments, the MYC portion of the MYC fusion peptide comprisesa MYC polypeptide sequence comprising the sequence shown below:

(SEQ ID NO: 9) PLNVSFTNRNYDLDYDSVQPYFYCDEEENFYQQQQQSELQPPAPSEDIWKKFELLPTPPLSPSRRSGLCSPSYVAVTPFSLRGDNDGGGGSFSTADQLEMVTELLGGDMVNQSFICDPDDETFIKNIIIQDCMWSGFSAAAKLVSEKLASYQAARKDSGSPNPARGHSVCSTSSLYLQDLSAAASECIDPSVVFPYPLNDSSSPKSCASQDSSAFSPSSDSLLSSTESSPQASPEPLVLHEETPPTTSSDSEEEQEDEEEIDVVSVEKRQAPGKRSESGSPSAGGHSKPPHSPLVLKRCHVSTHQHNYAAPPSTRKDYPAAKRVKLDSVRVLRQISNNRKCTSPRSSDTEENVKRRTHNVLERQRRNELKRSFFALRDQIPELENNEKAPKVVILKKATAYILSVQAEEQKLISEKDLLRKRR EQLKHKLEQLR.

In some embodiments, the MYC fusion peptide comprises SEQ ID NO: 10; insome embodiments, the MYC-fusion peptide is SEQ ID NO: 10.

(SEQ ID NO: 10) MRKKRRQRRRPLNVSFTNRNYDLDYDSVQPYFYCDEEENFYQQQQQSELQPPAPSEDIWKKFELLPTPPLSPSRRSGLCSPSYVAVTPFSLRGDNDGGGGSFSTADQLEMVTELLGGDMVNQSFICDPDDETFIKNIIIQDCMWSGFSAAAKLVSEKLASYQAARKDSGSPNPARGHSVCSTSSLYLQDLSAAASECIDPSVVFPYPLNDSSSPKSCASQDSSAFSPSSDSLLSSTESSPQASPEPLVLHEETPPTTSSDSEEEQEDEEEIDVVSVEKRQAPGKRSESGSPSAGGHSKPPHSPLVLKRCHVSTHQHNYAAPPSTRKDYPAAKRVKLDSVRVLRQISNNRKCTSPRSSDTEENVKRRTHNVLERQRRNELKRSFFALRDQIPELENNEKAPKVVILKKATAYILSVQAEEQKLISEKDLLRKRREQLKHKLEQLRKGELNSKLEGKPIPNPLLGLDSTRTG HEIHHHH.

The fusion protein may be modified during or after synthesis to includeone or more functional groups. By way of example but not by way oflimitation, the protein may be modified to include one or more of anacetyl, phosphate, acetate, amide, alkyl, and/or methyl group. This listis not intended to be exhaustive, and is exemplary only. In someembodiments, the protein includes at least one acetyl group.

III. Methods of Using Formulations of the Present Technology

Compositions of the present technology (e.g., compositions comprising aMYC-containing polypeptide formulated as biologically active, stablenanoparticles) provide MYC activity, and are thus useful in vivo (e.g.,as an adjuvant, immune system enhancer, etc.) and in vitro (e.g., tostimulate growth and proliferation of stem cells, such as hematopoieticstem cells (HSCs), to condition HSCs for enhanced engraftment followinghematopoietic stem cell transplantation, induce or enhance activation,growth, proliferation, viability, or survival of an immune cell and/orenhance antibody production by an immune cell in culture, etc.).

By way of example only and not by way of limitation, MYC-containingnanoparticulate compositions of the present technology can be used toprime donor HSCs (e.g., the patient's isolated HSCs or third partydonor's HSC) for transplantation, e.g., to patients with immune-relateddiseases or disorders such as, but not limited to severe combinedimmunodeficiency.

Severe combined immunodeficiency (SCID) is a life-threatening primaryimmunodeficiency disease caused by defects compromising the quantity andfunction of T-cells and B-cells. Infants with SCID suffer from repeatedlife-threatening infections that usually lead to a diagnosis by the ageof three to six months. Left untreated, children continue to experiencesevere, life-threatening infections and death by 2 years of age.Children with SCID are currently treated using HSCT designed toreconstitute a normal immune system. Outcomes of these transplants arebest for the youngest infants, who are transplanted before they havesuffered from a severe infection (most of these infants are eithersiblings of a proband or are identified by neonatal screening programs)and for patients with human leukocyte antigen (HLA)-matched familydonors. In contrast, the results of transplants performed on olderinfants and those performed with cells from alternative donors are lessfavorable.

In some embodiments, T-cell and B-cell depleted donor hematopoieticcells, including hematopoietic stem and progenitor cells (HSPCs), areincubated ex vivo for one with the MYC-containing nanoparticulatecompositions of the present technology (“primed”). Following incubation,the primed cells are washed and transplanted into the patient.Incubation of the donor cells with the compositions of the presenttechnology increased proliferation of long-term self-renewinghematopoietic stem cells following transplantation.

In some embodiments, the donor hematopoietic cells are isolated from thepatient. In some embodiments, the donor hematopoietic cells areincubated for 30 minutes, 60 minutes, 90 minutes or 120 minutes with thenanoparticulate MYC-containing compositions of the present technology.In some embodiments, the donor hematopoietic cells are incubated with 10μg/ml, 20 μg/ml, 30 μg/ml, 40 μg/ml, 50 μg/ml, 60 μg/ml, 70 μg/ml, 80μg/ml, 90 μg/ml, or 100 μg/ml of the nanoparticulate MYC-containingcomposition of the present technology. In some embodiments, cells areincubated with 50 μg/ml of a nanoparticulate MYC-containing compositionsfor 60 minutes. In some embodiments, the nanoparticles of thecomposition have an average particle size of between about 80 and 150nm, between about 90 and 140 nm, between about 100 and 120 nm, orbetween about 100 and 110 nm, and comprise SEQ ID NO: 1. In someembodiments, the nanoparticles of the composition have an averageparticle size of between about 80 and 150 nm, between about 90 and 140nm, between about 100 and 120 nm, or between about 100 and 110 nm, andcomprise SEQ ID NO: 10.

For in vivo use, in some embodiments, pharmaceutical formulationsincluding the nanoparticulate MYC-containing proteins described hereinand optionally one or more additional therapeutic compounds andoptionally, one or more pharmaceutically acceptable excipients, areadministered to an individual in any manner, including one or more ofmultiple administration routes, such as, by way of non-limiting example,oral, parenteral (e.g., intravenous, intraperitoneal, subcutaneous,intramuscular), intranasal, buccal, topical, rectal, or transdermaladministration routes. The pharmaceutical formulations described hereininclude, but are not limited to, aqueous liquid dispersions,self-emulsifying dispersions, solid solutions, liposomal dispersions,aerosols, solid dosage forms, powders, immediate release formulations,controlled release formulations, fast melt formulations, tablets,capsules, pills, delayed release formulations, extended releaseformulations, pulsatile release formulations, multiparticulateformulations, and mixed immediate and controlled release formulations. Asummary of pharmaceutical formulations is found, for example, inRemington: The Science and Practice of Pharmacy, Nineteenth Ed (Easton,Pa.: Mack Publishing Company, 1995); Hoover, John E., Remington isPharmaceutical Sciences, Mack Publishing Co., Easton, Pa. 1975;Liberman, H. A. and Lachman, L., Eds., Pharmaceutical Dosage Forms,Marcel Decker, New York, N.Y., 1980; and Pharmaceutical Dosage Forms andDrug Delivery Systems, Seventh Ed. (Lippincott Williams & Wilkins 1999).

In some embodiments, the nanoparticle formulations provided hereincomprise a suitable buffer. Exemplary buffers include, but are notlimited to Tris, sodium phosphate, potassium phosphate, histidine orcitrate based buffers. In some embodiments, the formulations providedherein contain magnesium. In some embodiments, the formulations providedherein contain one or more surfactants. As used herein, the term“surfactant” can include a pharmaceutically acceptable excipient whichis used to protect protein formulations against mechanical stresses likeagitation and shearing. Examples of pharmaceutically acceptablesurfactants include polyoxyethylensorbitan fatty acid esters (Tween),polyoxyethylene alkyl ethers (Brij), alkylphenylpolyoxyethylene ethers(Triton-X), polyoxyethylene-polyoxypropylene copolymer (Poloxamer,Pluronic), and sodium dodecyl sulphate (SDS). Suitable surfactantsinclude polyoxyethylenesorbitan-fatty acid esters such as polysorbate20, (sold under the trademark Tween 20®) and polysorbate 80 (sold underthe trademark Tween 80®). Suitable polyethylene-polypropylene copolymersare those sold under the names Pluronic® F68 or Poloxamer 188®. SuitablePolyoxyethylene alkyl ethers are those sold under the trademark Brij®.Suitable alkylphenolpolyoxyethylene esthers are sold under the tradenameTriton-X. When polysorbate 20 (Tween 20®) and polysorbate 80 (Tween 80®)are used they are generally used in a concentration range of about 0.001to about 1%, of about 0.005 to about 0.2% and of about 0.01% to about0.1% w/v (weight/volume).

In some embodiments, the nanoparticle formulations provided hereincomprise a stabilizer. As used herein, the term “stabilizer” can includea pharmaceutical acceptable excipient, which protects the activepharmaceutical ingredient and/or the formulation from chemical and/orphysical degradation during manufacturing, storage and application.Chemical and physical degradation pathways of protein pharmaceuticalsare reviewed by Cleland et al., Crit. Rev. Ther. Drug Carrier Syst.,70(4):307-77 (1993); Wang, Int. J. Pharm., 7S5(2): 129-88 (1999); Wang,Int. J. Pharm., 203(1-2): 1-60 (2000); and Chi et al, Pharm. Res.,20(9): 1325-36 (2003). Stabilizers include but are not limited tosugars, amino acids, polyols, cyclodextrines, e.g.,hydroxypropyl-beta-cyclodextrine, sulfobutylethyl-beta-cyclodextrin,beta-cyclodextrin, polyethylenglycols, e.g., PEG 3000, PEG 3350, PEG4000, PEG 6000, albumin, human serum albumin (HSA), bovine serum albumin(BSA), salts, e.g., sodium chloride, magnesium chloride, calciumchloride, chelators, e.g., EDTA as hereafter defined. As mentionedhereinabove, stabilizers can be present in the formulation in an amountof about 10 to about 500 mM, an amount of about 10 to about 300 mM, orin an amount of about 100 mM to about 300 mM.

In some embodiments, the daily dosages for a composition includingfusion peptide nanoparticles described herein are from about 0.001 to1000.0 mg/kg per body weight, such as about 0.01 to 100.0 mg/kg per bodyweight, such as about 0.1 to 10.0 mg/kg per body weight. The foregoingrange is merely suggestive, as the number of variables in regard to anindividual treatment regime is large, and considerable excursions fromthese recommended values are not uncommon. In some embodiments, suchdosages are optionally altered depending on a number of variables, notlimited to the activity of the agent or composition described hereinused, the disorder or condition to be treated, the mode ofadministration, the requirements of the individual subject, the severityof the disorder or condition being treated, and the judgment of thepractitioner.

Toxicity and therapeutic efficacy of such therapeutic regimens areoptionally determined in cell cultures or experimental animals,including, but not limited to, the determination of the LD₅₀ (the doselethal to 50% of the population) and the ED₅₀ (the dose therapeuticallyeffective in 50% of the population). The dose ratio between the toxicand therapeutic effects is the therapeutic index, which is expressed asthe ratio between LD₅₀ and ED₅₀. An agent or compositions describedherein exhibiting high therapeutic indices is preferred. The dataobtained from cell culture assays and animal studies are optionally usedin formulating a range of dosage for use in human. The dosage of such anagent or composition described herein lies preferably within a range ofcirculating concentrations that include the ED₅₀ with minimal toxicity.The dosage optionally varies within this range depending upon the dosageform employed and the route of administration utilized.

VII. EXAMPLES

The following examples are for illustrative purposes only and arenon-limiting embodiments. Many modifications, equivalents, andvariations of the present disclosure are possible in light of the aboveteaching, therefore, it is to be understood that within the scope of theappended claims, the disclosure may be practiced other than asspecifically described.

Example 1: Construction of a TAT-MYC Fusion Peptide of the PresentTechnology

Plasmid pTAT-MYC-V5-6xHis was made by PCR amplification of the codingregions for human MYC using a forward primer that contains an in-frameN-terminal 10-amino-acid sequence of the TAT protein transduction domainof HIV-1 (MRKKRRQRRR (SEQ ID NO: 7), and a reverse primer that removesthe stop codon. The PCR product was cloned into pET101/D-Topo(Invitrogen) vector, which includes a C-terminal V5 epitope tag and6-histidine protein tags.

A. Bacterial Strain Used for Protein Expression

BL-21 RARE cells were created by transforming BL-21 Star™ E. coli strain(Invitrogen) with pRARE (CamR), isolated from BL21 Rosetta cells(Novagen), that express tRNAs for AGG, AGA, AUA, CUA, CCC, GGA codons.

B. Protein Induction and Purification

To prepare the fermenter inoculum, vials of the TAT-MYC Master Cell Bank(MCB) were thawed and used to inoculate shaker flasks containing LBmedia and supplemented with antibiotics (40 μg/mL kanamycin) forselection. Cells were expanded in an orbital shaker/incubator forfourteen to sixteen hours. The expanded culture was then used toinoculate the fermenter culture. The fermentations were induced at 37°C. by adding IPTG (˜0.5-1 mM) to the culture when the cells were inlogarithmic growth phase (OD of about 4.0). The cells were induced untilthe DO (dissolved oxygen) spike was observed, approx. 3-6 hours. Afterinduction, the cell paste was harvested by centrifugation and stored at−70° C. or below until further processing. The bulk of the proteincontained in the inclusion bodies is TAT-MYC.

The cell paste was resuspended in 8M Urea, 50 mM Phosphate pH 7.5. Thesuspension was mixed at room temperature until homogenous. Thesuspension was then passed through a homogenizer. The homogenizedsuspension was then adjusted to include 200 mM Sodium Sulfite and 10 mMSodium Tetrathionate. The solution was mixed at room temperature untilhomogeneous. The sulfonylated lysate was mixed at 2-8° C. for ≥12 hours.The sulfonylated lysate was then centrifuged for an hour. Thesupernatant was collected and the pellet discarded. The supernatant waspassed through a 0.22 μm membrane filter.

The sulfonylated TAT-MYC solution was purified by Ni affinitychromatography using Ni-resin. The column was equilibrated in 6M Urea,50 mM Phosphate, 500 mM NaCl, and 10% Glycerol solution. Thesulfonylated and clarified TAT-MYC was then loaded onto the column. Thecolumn was washed with 6M Urea, 50 mM Phosphate, 10% Glycerol, 500 mMNaCl, pH 7.5. The column was then washed with 6M Urea, 50 mM Phosphate,10% Glycerol, and 2M NaCl, pH 7.5, followed another wash of 6M Urea, 50mM Phosphate, 10% Glycerol, 50 mM NaCl, and 30 mM Imidazole, pH 7.5. Theproduct was eluted from the column by running elution buffer containing6M Urea, 50 mM Phosphate, 10% Glycerol, and 50 mM NaCl, pH 7.5 with agradient from 100 to 300 mM Imidazole and collecting fractions. Theprotein containing fractions to be pooled was filtered through a 0.22 μmmembrane and protein concentrations were measured using UV280.

The pooled fractions from the Ni-Sepharose chromatography step werefurther purified by anion exchange chromatography using Q-Sepharoseresin. The pool was prepared for loading onto the column by diluting tothe conductivity of the Q sepharose buffer (17.52+/−1 mS/cm) with thesecond wash buffer (6M Urea, 50 mM Phosphate, 10% Glycerol, 2M NaCl, pH7.5) from the Ni Sepharose chromatography step. The diluted pool wasthen loaded onto the column, followed by two chase steps using 6M Urea,50 mM Phosphate, 300 mM NaCl, and 10% Glycerol, and further chase untilthe UV trace reached baseline.

Example 2. Preparation of Nanoparticulate TAT-MYC Compositions

Refolding of the TAT-MYC proteins in the Q-Sepharose flow-through poolfrom Example 1 was accomplished using tangential flow filtration-basedrefolding method using a UFDF (ultrafiltration/diafiltration) membrane.The refolding process included a series of three refolding steps. Thefirst refolding step involved the exchange of refold buffer 1 (3M Urea,50 mM Phosphate, 500 mM NaCl, 10% Glycerol, 5 mM GSH, (ReducedGlutathione) 1 mM GSSG (Oxidized Glutathione)) over the course of about120 minutes. The second refold step involved the exchange of refoldbuffer 2 (1.5M Urea, 50 mM Phosphate, 500 mM NaCl, 10% Glycerol, 5 mMGSH, (Reduced Glutathione) 1 mM GSSG (Oxidized Glutathione)) over thecourse of approximately 120 minutes, followed by ˜120 minutes ofrecirculation. The third refold step consisted of the exchange of refoldbuffer 3 (50 mM Phosphate, 500 mM NaCl, 10% Glycerol, 5 mM GSH, (ReducedGlutathione) 1 mM GSSG (Oxidized Glutathione)) over the course ofapproximately 120 minutes, followed by 12 hours of recirculation. Afterthe third refolding step was complete, the refold 3 solution wasfiltered through a 0.2 μm membrane and protein concentration wasadjusted.

The protein concentration of the TAT-MYC fusion of SEQ ID NO: 1 wasmeasured by Bradford protein assay (Sigma) compared to a standard curveof bovine serum albumin.

Several lots of refolded TAT-MYC were prepared according to thedescribed method. For the purposes of these Examples, the lots presentedare termed “F01,” “C2A,” C2B,” “C6” “C7” “C12”, “C13” and “C14.” Eachtested lot contained 50 mM Phosphate, 500 mM NaCl, 10% Glycerol, 5 mMGSH, (Reduced Glutathione) 1 mM GSSG (Oxidized Glutathione) as obtainedfollowing the third refold step with refold buffer 3, with the exceptionof C2A formulation, which additionally contained arginine.

Refolded TAT-MYC is sensitive to pH and was stable in a formulation ofpH 7.5+/−pH 0.3. Refolded TAT-MYC is also sensitive to NaClconcentrations and was stable at about 500 mM+/−50 mM NaCl. RefoldedTAT-MYC was also stable up to a concentration of 1.2 mg/mL. RefoldedTAT-MYC is stable at −80° C. for up to or about 2 years. Once thawed, itremains active when stored at 4° C. for up to or about a month.

Example 3. Functional Analysis of Nanoparticulate TAT-MYC Compositions

TAT-MYC compositions were tested for activity as follows.

A spleen was harvested from a C57BL/6j (Jackson) mouse, and mechanicallydissociated through wire mesh. The red blood cells were removed, CD4positive T cells were isolated using commercially available isolationprocess (Dynabead), and the T cells were activated with 1 μg/ml anti-CD3and anti-CD28 antibody. The cells were plated into a 48 well clusterdish at 1.5×10⁶ cells per well in 1 ml of media. 24 hours later, theTAT-MYC formulation was added to the cells (a total of 12 μg per well)and incubated for 24 hours. 24 hours after adding the protein, the mediawas replaced and the cells were incubated for 48 hr. Cells were assessedfor viability at 96 hours after the initial activation via flowcytometry (forward×side scatter). Results for C2A (prepared according toExample 2) are shown in FIG. 1.

FIG. 1A (the plot on the left) shows the proliferation of untreated,activated T-cells (12.8). FIG. 1B (the plot on the right) shows theproliferation of activated T-cells treated with 6 μg of TAT-MYCformulation C2A for 24 hours. As shown, proliferation of T-cells morethan doubles with C2A treatment.

For purposes of these examples, an active preparation, composition,formulation, or fraction of TAT-MYC is one that provides at least a2-fold increase in T-cell proliferation as compared to control T-cells.

TABLE 1 Ratio of TAT-MYC Treated: Sample Nontreated Positive Control 2.0R147 1.1 R149 1.5 C12 2.0 C13 2.7

Example 4: Characterization of Nanoparticle Formulations

Compositions of nanoparticulate TAT-MYC protein, prepared as describedin Example 2 and tested for biological activity as described in Example3, were characterized using several diverse techniques to demonstratethat (a) TAT-MYC protein prepared by the methods of the presenttechnology surprisingly and unexpectedly forms nanoparticles having adiscreet size range; (b) only a sub-fraction of the nanoparticles withinthis range are biologically active, e.g., have MYC activity; and (c)activity is linked to both particle size and one or morepost-translational modifications.

A. Stability of Functional Formulations

Two different preparations of TAT-MYC protein, termed F01 and F02, wereevaluated by Asymmetrical Flow Field-flow Fractionation (AF4) andMulti-Angle Laser Light Scattering (MALLS) at two different temperaturesto provide information on mass and size distribution of all componentsin the sample. F01 was refolded according to Example 2. F02 was preparedas discussed below. The AF4-MALLS analysis illustrated that F01 includesprimarily a single population of particles having a discreet size range,and that particle size is stable over time and at varying temperature.

As noted above, F01 was prepared according to the methods of Example 2.F02 was prepared as described in Example 1B. After the refold, F02 wasmoved for the F01 formulation, 50 mM Phosphate, 500 mM NaCl, 10%Glycerol, 5 mM GSH, (Reduced Glutathione) 1 mM GSSG (OxidizedGlutathione), pH 7.5, to the final formulation of 50 mM Phosphate, 250mM NaCl, 10% Glycerol, pH 7.0.

FIG. 2A shows the results for F01. Four different samples are shown inFIG. 2A: 2 F01 samples that were run at 25° C. and 2 F01 samples thatwere run at 5° C. are presented. As shown in FIG. 2A, a single primarypeak of nearly identical relative scale is seen at about 24 to about 32minutes for all samples at both temperatures. This is indicative of acomposition comprising a stable population of particles of discreetsize.

FIG. 2B provides results for F02. Again, four different samples areshown: 2 F02 samples run at 25° C. and 2 F02 samples run at 5° C. Incontrast to the F01 results, several peaks were identified at varioustime points, all differing in relative scale. For peaks appearingbetween 10 and 20 minutes, the top trace and the third trace representresults from the samples run at 25° C.; the second and fourth tracerepresent the samples run at 5° C. For peaks appearing between 24minutes and 32 minutes, the top three traces include both 5° C. samplesand one of the 25° C. samples, while the lowest trace in this time framerepresents the second sample run at 25° C. The peaks at the 40-minutetime point show one of the 5° C. samples as the lowest trace, with theremaining three sample traces above.

While F01 showed function in the T-cell assay as described in Example 3,F02 did not.

Accordingly, formulations of the present technology comprise stable,TAT-MYC particles of discreet size, and have biological function.

B. Biological Activity of the Formulations of the Present Technology isLinked to Particle Size

-   -   1. Size Exclusion Chromatography and High Performance Liquid        Chromatography Verify Discreet Particle Sizes are Present in        Biologically Active Tat-Myc Preparation

To verify that the function of TAT-MYC protein is linked tonanoparticles of discreet size, three different preparations of TAT-MYCwere evaluated. Two functional preparations (termed “C12” and “C13”),and one non-functional preparation (“R147”), were characterized via sizeexclusion chromatography followed by high performance liquidchromatography as follows.

Functional C12 and C13 were made according to the procedures outline inExample 2. R147 was generated during a run where the TFF refold stepswere accelerated to investigate the necessity to have the refold stepstake 120 min, 120 min and 14 hours as detailed under Example 1B.Instead, the entire refold was achieved in 60 min.

SEC-HPLC procedure to analyze TAT-MYC was carried out on an Agilent 1100Series. 3 columns were configured in tandem. Setup was as follows: Guardcolumn—2000 A-500 A-300 A. The mobile phase was 50 mM Sodium Phosphate,500 mM NaCl pH 7.0 with a Flow rate of 1.0 ml/min, length of each runwas 40 min, and the protein was introduced onto the column in a 100 μLinjection volume.

The traces of the three samples are shown in FIG. 3. SEC-HPLC allows forhigh resolution of particle size distribution and shows two distinctpeaks at about 13 and 16 minutes. While the peak at 13 minutes hadactive particles, the peak at 24 minutes includes active particles. Notethat the peaks observed at 22-24 min are a result of excipients in therefold buffer.

-   -   2. Size Exclusion Chromatography and Multi-Angle Static Light        Scattering Analysis to Determine Particle Size

SEC-multi-angle static light scattering (MALS) was also used to analyzethe same three samples, to confirm particle size (molecular weight) tocontrol for and eliminate any unexpected molecular interactions withcolumns.

Results are shown in FIGS. 4A and 4B. FIG. 4A is a graph showingrefractive index (hatched lines) and molecular weight (solid lines) ofsample C2A formulated in 50 mM Phosphate, 500 mM NaCl, 10% Glycerol, 5mM GSH, (Reduced Glutathione) 1 mM GSSG (Oxidized Glutathione), 250 mMArg, pH 7.5. FIG. 4B is the SEC trace of C2A showing relative signal ofC2A refold buffer with glutathione, but no Arg (Active Sample 1), andrefold buffer with Arg, but no glutathione (Active Sample 2).

Results shown in FIG. 4A and 4B confirm that the majority of activeparticles have a molecular mass of about 10⁷-10⁹ daltons (see fractionat retention volume of about 12.5 mL).

-   -   3. Dynamic Light Scattering Analysis to Verify Particle Size

Biologically active formulation C2A and three additional preparations,C12, C13 (both active), and R149 (inactive) assayed to determineparticle size. Functional C12 and C13 were made according to theprocedures outline in Example 2. R149 was generated during a run similarto C12 and C13, but the TFF refold steps were replaced by dialysis thatwas conducted over a period of 6 hours. Functional C2A was madeaccording to the procedures outline in 0080-0082, but the finalformulation buffer included 250 mM Arg.

The sample were evaluated using Dynamic Light Scattering (DLS), atechnique that can be used to determine the size distribution profile ofsmall particles in suspension or polymers in solution. DLS measures thediffusion of particles moving under Brownian motion and converts thediffusion coefficient to hydrodynamic diameter using the Stokes-Einsteinrelationship:

$D_{h} = \frac{k_{B}T}{3{\pi\eta}\; D}$

where D_(h)=hydrodynamic diameter, k_(B)=Boltzmann's constant,

=dynamic viscosity, D=translational diffusion coefficient, andT=thermodynamic temperature.

Samples and standards were analyzed on a Malvern Zetasizer Nano (s).Test samples and controls were diluted in formulation buffer to aconcentration of 0.5 mg/mL prior to analysis. Samples were analyzed andsizing evaluated by intensity, number counts, and volume distribution.The reported value for the analysis was obtained from the intensityZ-Ave (d.nm) value.

Results are shown in FIGS. 5A-5D. FIG. 5A shows the DLS trace for C2A;the average particle size (diameter) was 106.2 nm.

FIGS. 5B-5D show DLS trace data for preparations R149 (inactive), C12(active) and C13 (active) respectively. The inactive preparation has anaverage particle size of 63 nm, while preparations C12 and C13 haveaverage particle sizes of 106 and 103 nm, respectively.

-   -   4. Nanoparticle Tracking Analysis to Verify Size Distribution of        Biologically Active Tat-Myc Particles in Solution

Nanoparticle Tracking Analysis (NTA) is a method for visualizing andanalyzing particles in liquids that relates the rate of Brownian motionto particle size. The rate of movement is related only to the viscosityand temperature of the liquid; it is not influenced by particle densityor refractive index. NTA allows the determination of a size distributionprofile of small particles with a diameter of approximately 10-1000nanometers (nm) in liquid suspension.

Nanoparticle size and concentrations were measured by nanoparticletracking analysis (NTA) with a NS300 instrument (Malvern, Worchester,UK) equipped with a 488-nm laser and NTA 2.3 software. Before dataacquisition, the sample was diluted 1:200-1:400 in a final volume of 400μL and was loaded into the flow cell. Video was captured at roomtemperature for 60 s. The lower size detection limit was automaticallyset by the software.

Results are shown in FIGS. 6A and 6B. NTA analysis was performed intriplicate with the active C2A preparation. FIG. 6A indicates that themajority of particles in the sample are about 100 nm in size. FIG. 6Bshows the averaged concentration and size. A statistical breakdown ofparticle size in the sample is provided in the table below.

Mean +/− standard Merged Data Particle Size error Particle Size Mean116.0 nm Mean 116.9 +/− 4.7 nm Mode  98.5 nm Mode 101.5 +/− 4.9 nm SD 48.7 nm SD  48.5 +/− 3.5 nm D10  71.7 nm D10  72.4 +/− 2.7 nm D50  98.6nm D50 100.1 +/− 4.1 nm D90 171.2 nm D90 174.4 +/− 9.0 nm

-   -   5. Electron Microscopy to Visually Verify Uniformity and Size of        Biologically Active Nanoparticulate Tat-MyC

The above biochemical results were further confirmed by electronmicroscopy. A TAT-MYC formulation prepared as described in Example 2 andtermed “C2B” was tested in the T-cell assay of Example 3 to verifybiological activity. C2B was visualized via electron microscopy asfollows.

Fifty-microliter drops of TAT-MYC nanoparticle formulation were spottedonto parafilm and then absorbed to glow-discharged, carbon- andformvar-coated copper transmission electron microscopy (TEM) grids (G400copper; EM Science). Grids were blotted dry on Kimwipe (Kimberly-Clark),followed by staining with uranyl acetate (2% [wt/vol]) and TEM (PhilipsCM10) at 80 kV. The particles were observed at ×4,800 magnification.

Results are shown in FIG. 7. FIG. 7 shows discreet particles in the 100nm size range (dark balls). The particles are uniform in shape, andwhile some variation in size was noted in the micrograph, the data insections B-E confirm that the size range of active particles is inagreement with those shown in the micrograph.

C. Biological Function of the TAT-MYC Formulations of the PresentTechnology

To further distinguish active versus inactive TAT-MYC preparations,peptide mapping was performed. Peptide mapping, also known as peptidefingerprinting, is a technique of forming two-dimensional patterns ofpeptides (on paper or gel) by partial hydrolysis of a protein followedby electrophoresis and chromatography. The peptide pattern (orfingerprint) produced is characteristic for a particular protein and thetechnique can be used to separate a mixture of peptides.

Six different TAT-MYC formulations were digested with endoprotease AspN.Three of the samples C2B, C6, and C7, were prepared according to Example2 and showed biological activity in the T-cell assay of Example 3. Threeof the samples, C4, 147, and 149, were prepared as follows. R149 wasgenerated during a run similar to C12 and C13, but the TFF refold stepswere replaced by dialysis that was conducted over a period of 6 hours.R147 was generated during a run were the TFF refold steps wereaccelerated to investigate the necessity to have the refold steps take120 min, 120 min and 14 hours as detailed under Example 2. Instead, theentire refold was achieved in 60 min. C4 was made according to theprocedures outlined in Example 1B, but rather then adjusting the saltand conductivity of the Q load for a flow through, this Q column was runas a bind and then eluted over a salt gradient. None of these samplesshowed biological activity in the Example 3 T-cell assay.

Protein digests were performed as follows. TAT-MYC was denatured byguanidine hydrochloride, reduced by TCEP (Tris 2-carboxyethylphosphine), alkylated by iodoacetamide, and digested by endoproteinaseAsp-N, which cleaves at the N-terminal of the aspartic acid residues.The resulting peptides were separated by reversed phase HPLC andmonitored with 215 nm absorbance. Peptide identity in the peak elutionprofile was established by mass spectrometry. For routine proteinanalysis, identity may be established by visual comparison of the peakprofiles in the UV-chromatogram of the sample to a reference sample.

Protein fragments were analyzed by HPLC. Results are shown in FIG. 8.The top three traces of FIG. 8 represent the biologically activesamples. The bottom three traces represent the inactive samples. Asshown in FIG. 12, a peak between the 12 and 12.5 minute time points,immediately to the right of the peak labeled A1, A1-2, was present inall three active samples, but was absent or minimally visible in theinactive samples. Without wishing to be bound by theory, it is likelythat one or more aspartic acid residues is acetylated in the activesample.

Example 5: Structural Characterization of MYC-Containing NanoparticleFormulations

The primary, secondary, tertiary and quaternary structures ofMYC-containing nanoparticle formulations were evaluated using an arrayof biochemical, biophysical and functional characterization techniquesas outlined in the table below using standard techniques.

Summary of MYC-Containing Nanoparticle Structure and FunctionElucidation Strategy

Structure Quality Attribute Assay Primary Structure Post translationalPeptide Mapping modifications RP-HPLC Secondary/Tertiary FoldingPatterns Far-UV CD Spectroscopy Structure Near-UV CD Spectroscopy FTIRSpectroscopy Quaternary Structure High Molecular AnalyticalUltracentrifugation Weight Species DLS dSEC-HPLC Higher Order ElectronMicroscopy Structure

Nanoparticle formulation C13 was used for the majority ofcharacterization analyses presented in this example. In some cases, datafrom an alternate nanoparticle formulation (either C2B or C14) ispresented herein. Nanoparticle formulation C14 was also produced at asimilar manufacturing scale according to essentially the same process aswas utilized for nanoparticle formulation C13.

A. Peptide Mapping by Liquid Chromatography—Mass Spectrometry (LC/MS)

The myc-containing nanoparticle formulations were analyzed as describedin the chart above with the addition of mass spectrometry withElectrospray Ionization (ESI) with a Time of Flight (TOF) analyzer.Because there was no UV detection during the MS run, a visual alignmentof the MS Total Ion Chromatogram (TIC) and the UV chromatogram wasperformed to assign the observed mass to the respective UV peaks.

The mass spectrometry analysis confirmed 100% coverage of peptides basedon the expected peptide sequence. FIG. 9 shows a representative peptidemap for MYC peptides with peptide peaks identified. The assignment ofpeptides for each peak is provided in the table below.

Theoretical TAT-MYC Asp-N Peptides

Peptide Amino Acid # Position Sequence 1  1-21(−)MRKKRRQRRRPLNVSFTNRNY(D) (SEQ ID NO: 12) 2 22-23 (Y)DL(D)(SEQ ID NO: 13) 3 24-25 (L)DY(D) (SEQ ID NO: 14) 4 26-34 (Y)DSVQPYFYC(D)(SEQ ID NO: 15) 5 35-56 (C)DEEENFYQQQQQSELQPPAPSE(D) (SEQ ID NO: 16) 657-93 (E)DIWKKFELLPTPPLSPSRRSGLCSPSYVAV TPFSLRG(D) (SEQ ID NO: 17) 794-95 (G)DN(D) (SEQ ID NO: 18) 8  96-105 (N)DGGGGSFSTA(D)(SEQ ID NO: 19) 9 106-117 (A)DQLEMVTELLGG(D) (SEQ ID NO: 20) 10 118-126(G)DMVNQSFIC(D) (SEQ ID NO: 21) 11 127-128 (C)DP(D) (SEQ ID NO: 22) 12129-129 (P)D(D) (SEQ ID NO: 23) 13 130-140 (D)DETFIKNIIIQ(D)(SEQ ID NO: 24) 14 141-166 (Q)DCMWSGFSAAAKLVSEKLASYQAARK(D)(SEQ ID NO: 25) 15 167-188 (K)DSGSPNPARGHSVCSTSSLYLQ(D) (SEQ ID NO: 26)16 189-198 (Q)DLSAAASECI(D) (SEQ ID NO: 27) 17 199-209 (I)DPSVVFPYPLN(D)(SEQ ID NO: 28) 18 210-220 (N)DSSSPKSCASQ(D) (SEQ ID NO: 29) 19 221-229(Q)DSSAFSPSS(D) (SEQ ID NO: 30) 20 230-259(S)DSLLSSTESSPQGSPEPLVLHEETPPTTSS (D)(SEQ ID NO: 31) 21 260-266(S)DSEEEQE(D) (SEQ ID NO: 32) 22 267-271 (E)DEEEI(D) (SEQ ID NO: 33) 23272-326 (I)DVVSVEKRQAPGKRSESGSPSAGGHSKPPH SPLVLKRCHVSTHQHNYAAPPSTRK(D)(SEQ ID NO: 34) 24 327-336 (K)DYPAAKRVKL(D) (SEQ ID NO: 35) 25 337-357(L)DSVRVLRQISNNRKCTSPRSS(D) (SEQ ID NO: 36) 26 358-387(S)DlEENVKRRTHNVLERQRRNELKRSFFALR (D)(SEQ ID NO: 37) 27 388-426(R)DQIPELENNEKAPKVVILKKATAYILSVQA EEQKLISEE(D) (SEQ ID NO: 38) 28427-464 (E)DLLRKRREQLKHKLEQLRKGELNSKLEGKP IPNPLLGL(D) (SEQ ID NO: 39) 29465-476 (L)DSTRTGHHHHHH(-) (SEQ ID NO: 40)

B. Reversed Phase High Performance Liquid Chromatography (RP-HPLC)

A reversed phase HPLC (RP-HPLC) method was employed for the quantitationof post translational modifications and product related impurities. Thetest sample was diluted in denaturing buffer (7.5 M Guanidine HCl,0.0625M TrisHCl pH 7.3) and separated using an Agilent AdvanceBio RP-mAbC4 column (2.1×150 mm, Solid core beads 3.5 μm, 450 Å). The column wasequilibrated at a temperature of 50° C. Elution was achieved by applyinga flow rate of 0.5 mL/min and a linear gradient of 0.1% TFA (w/v) in100% H₂O (Eluent A) and 0.1% (w/v) TFA in 100% ACN (Eluent B) as shownin the table below. Detection was performed at 280 nm.

RP-HPLC Gradient Profile

Time (min) % Eluent B 0.00 23.0 15.00 37.0 35.00 45.0 36.00 70.0 37.0070.0 38.00 23.0 45.00 23.0

Analysis of nanoparticle formulation C13 indicated the presence of themain peak and a few minor peaks, as illustrated in FIG. 10. Theidentification of impurity peaks was not performed in this particularexample.

C. Circular Dichroism Spectroscopy

The structure of nanoparticle formulation C13 was evaluated usingCircular Dichroism spectroscopy (CD) in both the “far-UV” spectralregion (190-250 nm) and the “near-UV” spectral region (250-350 nm). Inthe far UV region, the chromophore is the peptide bond and the signalarises when it is located in a regular, folded environment. The near UVregion can be sensitive to certain aspects of tertiary structure. Atthese wavelengths, the chromophores are the aromatic amino acids anddisulfide bonds, and the CD signals they produce are sensitive to theoverall tertiary structure of the protein.

Nanoparticle formulation C13 was diluted to an absorbance of 0.75 AU at280 nm for near UV, and 1.1 AU at 195 nm for far UV. Glutathione has asignificant impact of sample absorbance in the UV. Thus, to increase thesignal contribution from the protein, samples were buffer exchanged intoreduced glutathione and glutathione free buffers. Testing parameters forthe samples are shown in table below.

Testing Parameters for Characterization of Nanoparticle Formulations byCD Spectroscopy

Parameter Value Temperature Ambient Concentration Various concentrationtested Wavelength Range 340-240 nm for NUV 250-195 nm for FUV Bandwidth 1 nm Step size 0.5 nm Collection Interval 1 second Readings¹ 3 for NUV5 for FUV ¹All readings were averaged to produce the reported spectrumfor each sample

For both far and near UV CD spectroscopy, the averaged spectrum wasbuffer subtracted and normalized to the mean residue molar ellipticity(MRME). A quantitative analysis of spectral similarity was performedusing the weighted spectral difference (WSD) algorithm. The resultingfar UV spectra, provided in FIG. 11, suggest that protein secondarystructure consists primarily of β-sheets. There was little evidence ofα-helical structure present. The near UV spectra, provided in FIG. 12,indicated weak tryptophan, high tyrosine, and very high disulfidefeatures, This result is consistent with the number of tryptophan,tyrosine and disulfides residues in the molecule, as shown in the tablebelow.

Tryptophan, Tyrosine and Disulfides in the MYC-Containing NanoparticleFormulations

Amino Acid Residue Number per Molecule Tryptophan  2 Tyrosine 12Disulfides  4 (9 cysteines)

D. Fourier Transform Infrared Spectroscopy (FTIR)

Fourier transform infrared spectroscopy (FTIR) spectra from wavenumbers1700-1500 cm⁻¹ can be used to determine structural properties ofproteins. Analysis in this spectral region results in two absorptionbands, conventionally called Amide I and Amide II and lying betweenwavenumbers 1700-1600 cm⁻¹ and 1600-1500 cm⁻¹, respectively. The Amide 1band is due to C═O stretching vibrations of the peptide bonds, which aremodulated by the secondary structure (α-helix, β-sheet, etc.). Secondarystructural content can be obtained by comparing the measured spectra tothe spectra obtained for proteins with known secondary structures. FTIRspectroscopy for nanoparticle formulation C14 was performed using aBruker Optics Vertex 70, with MCT detector and BioATR Cell II. Thesamples required no preparation prior to analysis. Testing parametersfor the analysis are shown in in the table below.

FTIR Testing Parameters

Parameter Value Temperature 25° C. Concentration Neat Detector MCTAperture Setting 6 mm Resolution 4 cm⁻¹ Beamsplitter Setting KBr HighPass Filter Open Low Pass Filter 10 kHz Wavenumber Range* 6000-950 cm⁻¹Scanner Velocity 20 kHz Number of scans 128 *Wave number range wassuggested. Buffer subtraction is typically done between 2800-1000 cm⁻¹and secondary structure spectral analysis performed between 1700-1600cm⁻¹.

The resulting FTIR spectrum was buffer subtracted and min-max normalizedacross 1600 to 1700 cm⁻¹. Nine-point smoothing was applied to minimizewhite noise for the determination of the second derivative. Aquantitative analysis of spectral similarity was performed using theweighted spectral difference (WSD) algorithm.

Nanoparticle formulation C14 showed a significant beta structure, bothsheet and turn. The peak around 1649-1655 is random coil. These spectralfeatures are consistent with the qualitative analysis of the far UV CDdata: i.e. primarily beta structures and random coil, and very little tono α-helix is present.

E. Analytical Ultracentrifugation

The higher order structure of nanoparticle formulation C13 wasinvestigated based on the sedimentation behavior of the protein insolution by sedimentation velocity analytical ultracentrifugation(SV-AUC). SV-AUC measures the rate at which molecules sediment inresponse to a centrifugal force. This sedimentation rate providesinformation on the molecular weight of molecules present in the sample.

SV-AUC was performed using a Beckman-Coulter XLI, using absorbanceoptics. The samples required no additional preparation prior toanalysis. The testing parameters are shown in the table below. Dataanalysis was performed using SEDFIT 15.01b using parameters as shown inthe table below. AUC data are illustrated in FIG. 14.

AUC Testing Parameters

Parameter Value Temperature 20° C. Concentration Neat DetectionAbsorbance at 280 nm Angular Velocity 20,000 rpm Radial scan increment0.003 cm Centerpiece Epon 12 mm, 2 sector

AUC Data Analysis Parameters

Parameter Value Meniscus Float from max of sample side spike Bottom 7.2cm Bottom fitting limit 6.7 cm Frictional ratio Float from 2.2 Bufferdensity 1.05507 g/L Buffer Viscosity 0.01628 Poise Partial specificvolume 0.73 Sedimentation coefficient range 0.1¹-500 S RegularizationMethod Tikhonov-Phillips Time independent noise Enabled Radialindependent noise Disabled ¹Log spaced s grid was used thus all valuesmust be greater than zero

Nanoparticle formulation C13 demonstrated a large molecular weightdistribution. Approximate molecular weights were determined using knownbuffer properties. The distribution began sharply at ˜4 MDa (20.6 S) andhad an apex at ˜10 MDa (40 S). The main population distribution extendedout to ˜120 MDa (185 S). Low quantities of larger species extendedbeyond 400 S, however these species are below the limit of quantitationof the method and cannot be accurately quantified.

F. Denaturing Size Exclusion Chromatography-HPLC

Nanoparticle formulation C13 was analyzed by denaturing size exclusionchromatography-HPLC (dSEC-HPLC). The samples were run under denaturingconditions, both reducing and non-reducing conditions utilizing a TSKgelG3000SWx1; 5 μm; A Stainless Steel 7.8 mm×30 cm column was used toachieve size-based separation of monomer from larger molecular weightspecies within the sample.

For the analysis of samples under non-reducing conditions, elution wasachieved using 7.5M Gdn HCl, 0.01M Sodium Acetate pH 4.7 as mobile phasein an isocratic configuration. Samples were diluted 1/10 in mobile phaseand vortexed for 10 seconds to mix. The samples were then incubated at37° C. for 30 minutes before placing into the autosampler at 4° C.

For the analysis of samples under reducing conditions, elution wasachieved using 7.5M Gdn HCl, 0.0625M TrisHCl pH 7.3 as mobile phase inan isocratic configuration. Samples were diluted 1/10 in mobile phase,and 1 mL of TCEP was added prior to vortexing for 10 seconds to mix. Thesamples were then incubated at 37° C. for 15 minutes before placing intothe autosampler at 4° C.

Detection for both reducing and non-reducing conditions was performedusing fluorescence for quantitation of peaks. An evaluation of sizedistribution across peaks was achieved using multi-angle laser lightscattering (MALLS).

The resulting chromatograms for SEC-HPLC analysis of nanoparticleformulation C13 are shown in FIG. 15. The overlaid traces on eachchromatogram represent testing that was performed approximately 1 monthapart for samples stored at 28° C., demonstrating the ability of themethod to pick up changes upon storage at accelerated temperature.

G. Electron Microscopy

Electron microscopy was performed for the collection of images of thethree dimensional structure of nanoparticle formulation C2B. Afifty-microliter drop of the nanoparticle formulation (100 ug/mL) wasspotted onto parafilm and then absorbed to glow-discharged, carbon- andformvar-coated copper transmission electron microscopy (TEM) grids (G400copper; EM Science). Grids were blotted dry on Kimwipe (Kimberly-Clark),followed by staining with uranyl acetate (2% [wt/vol]) and TEM (PhilipsCM10) at 80 kV. The number 100 -nm particles were imaged at ×4,800magnification. The resulting images confirm a highly ordered complex ofself-assembling spheres, as shown in FIG. 7.

Example 6: Characterization of Mutant TAT-MYC Peptide Formulations

In this Example, point mutations in MYC at sites that play a role in MYCfunction, were examined to determine whether the mutations affect thestability of a TAT-MYC nanoparticle formulation. As described above, aTAT-MYC fusion protein having the sequence set forth in SEQ ID NO: 1 isa fusion protein that combines the protein transduction domain (PTD) ofHIV-1 TAT fused in frame to the wild-type human MYC protein (c-MYC),followed by two tags: V5 and 6xHis. TAT-3AMYC is identical to TAT-MYC,except that it contains 3 amino acid substitutions: T358A, S373A, andT400A. These three amino acids were initially selected for mutationbecause phosphorylation at all three residues has been shown to reduceMYC function by inhibiting the association of MYC with DNA, either byblocking Max binding or by interfering directly with binding to DNA(Huang et al. Mol Cell Biol. 24(4): 1582-1594 (2004)). As detailedbelow, the point mutations of TAT-3AMYC appear to destabilize thecomplex formed render the fusion protein preparation non-functional.

Preparation of TAT-MYC and TAT-3AMYC Fusion Proteins

TAT-MYC and TAT-3AMYC proteins were prepared as described above inExamples 1 and 2. The fusion proteins were solubilized under denaturingconditions and then refolded into a final formulation containing salts,glycerol and reducing agent (i.e. 50 mM Phosphate, 500 mM NaCl, 10%Glycerol, 5 mM GSH, (Reduced Glutathione) 1 mM GSSG (OxidizedGlutathione).

Size Exclusion Chromatography HPLC (SEC-HPLC)

The nanoparticle formulations comprising TAT-MYC and TAT-3AMYC were thenanalyzed for their molecular weight profiles using size exclusionchromatography HPLC (SEC-HPLC). Size variants were separated with anisocratic mobile phase (4M GdnHCl, 10 mM NaOAc, pH 4.65) over a sizeexclusion column and were monitored on a Diode Array Detector withultraviolet (UV) detection at 280 nM. The parameters used were asfollows: Flow rate: 0.75 mL/min; Maximum pressure limit: 70.0 bar; Runtime: 30 minutes; Injection Volume: 20 μL).

FIG. 16A shows the chromatogram of the TAT-MYC protein preparation. Theprotein complex elutes between minutes 6 and 7. Smaller proteinmultimers and excipient peaks can be seen eluting between minutes 8 and15. FIG. 16B shows the chromatogram of TAT-3AMYC compared to the TAT-MYCprotein preparation. The TAT-3AMYC has significantly less proteincomplex that elutes between minutes 6 and 7. The bulk of the proteinpreparation was comprised of smaller protein multimers and excipientpeaks can be seen eluting between minute 8 and 17. This indicates thatmutation of T358, S373, and T400 to alanine are sufficient to disruptthe formation of the nanoparticle complex that elutes between minute 6and 7 on an SEC column.

Potency Assay

The TAT-MYC and TAT-3AMYC fusion proteins were also assessed for potencyby testing their ability to rescue activated CD4+ T-cells fromapoptosis, retain a blasting phenotype, and continue to proliferateafter the antigenic stimulation. T cells were for the assay wereobtained by harvesting Spleen and Lymph Nodes from mice. The spleen andlymph nodes were ground through the mesh screen to generate a singlecell suspension in C10 culture media. The cells were transferred to aconical tube and pelleted by centrifugation at 1200 RPM for 5 min. Thecells were resuspended in 5 ml sterile TAC buffer (135 mM NH₄Cl, 17 mMTris pH 7.65) and allowed to sit in TAC buffer for 1-2 min to lyse thered blood cells. The cells were centrifuged at 1200 RPM for 5 min,washed with 10 mls of C10 media, and resuspended in 4 ml C10 medium.

T cells were isolated from the resuspended RBC-depleted cell mixtureusing anti-CD4 Dynabeads according to the manufacturer's instructions(Invitrogen Cat #11445D). Briefly, 4 ml of the resuspended cell pelletwas added to a snap cap tube with 50 μl washed CD4 Dynabeads (×4) andincubated for 1 hour at 4° C. on a 360° Nutator. After incubating, thetubes were placed on a Dynal magnet, allowing two minutes for beads andcells to collect on the side of the tube. The supernatants (CD4 negativecells) were removed. The beads and bound cells were resuspended in 4 mlC10 media, and placed back on magnet to allow for separation. This washwas repeated. After washing, the beads and bound cells were resuspendedin 4 ml C10, and 50 μl CD4 detachabead (Invitrogen Cat #12406D) wasadded to each tube and incubated for 1 hour at 22° C. on a 360° Nutator.After incubating, the tubes were placed on the Dynal magnet, allowingtwo minutes for beads to collect on the side of the tube. Thesupernatants containing the CD4+ cells were collected in a 50 ml conicaltube. The remaining beads were resuspended in 10 ml C10 media, separatedon the Dynal magnet and collected. This step was then repeated and thesupernatants pooled. The pooled isolated CD4+ cells were centrifuged at1200 rpms for 5 minutes. The supernatant was removed and the cells wereresuspended in 20 ml (5 ml/mouse) at approximately 1.5×10⁶ cells/ml. 20ul commercial anti-CD3 (eBiosciences Cat #16-0031-86) and 20 ulanti-CD28 antibody (clone 37N1) at 1 ul/ml were added to the tube toactivate the T cells and the cells were seeded in 20 wells of 24 welldish, 1 ml of media per well and incubated 72 hr at 36° C.

At 72 hours post activation with anti-CD3 and anti-CD28, the media andthe cells were removed from each well from 24 well dish using media towash cells from the bottom of each well. The cells were pelleted at 1200rpms for five minutes, resuspended in 5 ml C10 medium and transferred to15 ml conical tube. The cells were underlayed with 5 ml Ficoll-Paque andspun at 1200 rpms, five minutes. The buffy coat was removed using a 4 mlglass pipette and transferred to a new 15 ml conical tube. 10 ml of C10media was added to wash cells. The cells were pelleted at 1200 rpms forfive minutes, resuspended at 1×10⁶ cells/ml and seeded 1 ml per well ofa 24-well plate. The cells were then treated with the TAT-MYC orTAT-3AMYC fusion proteins. Viability was determined by 48 hr aftertreating with fusion proteins using flow cytometry.

FIG. 17 shows a graphical representation of an activated T-cell potencyassay evaluating the ability of TAT-MYC and TAT-3AMYC to rescueactivated T-cell from apoptosis that follows cytokine withdrawal.TAT-MYC showed a 3 fold increase in the live T-cell population aftercytokine withdrawal compared to no treatment (NT). TAT-3AMYC, however,showed no increase the live population T-cell population compared to notreatment (NT). This result was in line with the chromatography data,which showed that the majority of the TAT-3AMYC does not form a complexlike TAT-MYC as shown in FIG. 16B.

In summary, TAT-MYC forms a nanoparticle complex that can be measured bySEC-HPLC and elutes between minute 6 and 7. TAT-3AMYC does not form thesame nanoparticle complex as TAT-MYC. Mutation of T358, S373, and T400to alanine was sufficient to disrupt the formation of the complex thatelutes between minutes 6 and 7 on an SEC column. Further, the functionof TAT-MYC observed in a T-cell potency assay appears to correlate withformation of this complex.

Example 7: Construction and Characterization of a TAT-MYC Fusion PeptideDerived from Chlorocebus sabaeus (green monkey) MYC protein Constructionand Purification of Green Monkey TAT-MYC

Plasmid pTAT-MYC (Green Monkey)-V5-6xHis was made by PCR amplificationof the coding regions for C. sabaeus (green monkey) MYC and replacingthe nucleic acid encoding human MYC sequence in the human TAT-MYC vectorof Example 1 with a portion of the green monkey MYC sequence encodingSEQ ID NO: 8, which differs from the human MYC sequence by two aminoacids. Protein production and purification were performed as describedin Example 1. Preparation of nanoparticulate TAT-MYC composition wasperformed as described in Example 2.

Dynamic Light Scattering (DLS) to Verify Particle Size

The refolded nanoparticulate compositions were assessed for nanoparticlesize distribution by Dynamic Light Scattering (DLS) technique asdescribed in Example 4(B)(3). Results are shown in FIG. 18, which showsthe DLS traces for the Green Monkey TAT-MYC nanoparticulatecompositions. The average particle size (diameter) for the Green MonkeyTAT-MYC was about 80 nm.

Reversed Phase High Performance Liquid Chromatography (RP-HPLC)

A reversed phase HPLC (RP-HPLC) method was employed for the quantitationof post translational modifications and product related impurities inthe Green Monkey TAT-MYC nanoparticulate composition. The test samplewas diluted in denaturing buffer (7.5 M Guanidine HCl, 0.0625M TrisHClpH 7.3) to 1 mg/ml. 2 μl of 0.5M TCEP ((tris(2-carboxyethyl)phosphine)was added to the sample, incubated at 37° C. for 30 minutes, then cooledto 2-8° C. The sample was stored at 2-8° C. for up to 5 days prior toanalysis. The sample (50 μl (˜5 μg) was separated using an AgilentAdvanceBio RP-mAb C4 column (2.1×150 mm, Solid core beads 3.5 μm, 450Å). The column was equilibrated at a temperature of 50° C. Elution wasachieved by applying a flow rate of 0.5 mL/min and a linear gradient of0.1% TFA (w/v) in 100% H₂O (Eluent A) and 0.1% (w/v) TFA in 100% ACN(Eluent B) as shown in the table below. Detection was performed at 215nm. Results for the Green Monkey TAT-MYC nanoparticulate compositioncompared to a reference human TAT-MYC nanoparticulate composition areshown in FIG. 19.

Size Exclusion Chromatography and High Performance Liquid Chromatography(SEC-HPLC)

The Green Monkey TAT-MYC nanoparticulate compositions were alsocharacterized via size exclusion chromatography followed by highperformance liquid chromatography. The samples were run underdenaturing, non-reducing conditions utilizing a TSKgel G3000SWx1; 5 μm;A Stainless Steel 7.8 mm×30 cm column to achieve size-based separationof monomer from larger molecular weight species within the sample.Elution was achieved using 4M Gdn HCl, 0.1M Sodium Acetate pH 4.65 asthe mobile phase in an isocratic configuration. Samples were diluted1/10 in mobile phase and vortexed for 10 seconds to mix. The sampleswere then incubated at 37° C. for 30 minutes before placing into theautosampler at 4° C.

The resulting chromatograms for SEC-HPLC analysis of Green MonkeyTAT-MYC nanoparticulate compositions compared to a reference humanTAT-MYC nanoparticulate composition are shown in FIG. 20.

Potency Assay

The Green Monkey TAT-MYC nanoparticulate compositions and referencehuman TAT-MYC nanoparticulate compositions were also assessed forpotency by testing their ability to rescue activated CD4+ T-cells fromapoptosis, retain a blasting phenotype, and continue to proliferateafter the antigenic stimulation. The compositions were assayed forbiological activity according to the potency assay described in Example6.

FIG. 21 shows a graphical representation of an activated T-cell potencyassay evaluating the ability of Green Monkey TAT-MYC and human TAT-MYCat various doses to increase the amount of activated T-cells having ablasting phenotype relative no treatment control.

While preferred embodiments of the present disclosure have been shownand described herein, it will be obvious to those skilled in the artthat such embodiments are provided by way of example only. Numerousvariations, changes, and substitutions will now occur to those skilledin the art without departing from the disclosure. It should beunderstood that various alternatives to the embodiments of thedisclosure described herein may be employed in practicing thedisclosure. It is intended that the following claims define the scope ofthe disclosure and that methods and structures within the scope of theseclaims and their equivalents be covered thereby.

All patents, patent applications, provisional applications, andpublications referred to or cited herein are incorporated by referencein their entirety, including all figures and tables, to the extent theyare not inconsistent with the explicit teachings of this specification.

Other embodiments are set forth within the following claims.

1.-18. (canceled)
 19. A method for the preparation of a population ofbiologically active nanoparticles comprising one or more MYC-containingpolypeptides, the method comprising: (a) solubilizing MYC-containingpolypeptides in a solubilization solution comprising a concentration ofa denaturing agent to provide solubilized MYC-containing polypeptides;(b) performing a first refolding step on the solubilized MYC-containingpolypeptides with a first refold buffer comprising about 0.35 to about0.65 the concentration of the denaturing agent of step (a) and about 100mM to about 1M alkali metal salt and/or alkaline metal salt for at leastabout 30 to 180 minutes to provide a first polypeptide mixture; (c)performing a second refolding step on the first polypeptide mixture witha second refold buffer comprising about 0.10 to about 0.30 theconcentration of the denaturing agent of step (b) and about 100 mM to 1Malkali metal salt and/or alkaline metal salt at least about 30 to 180minutes to provide a second polypeptide mixture; and (e) performing athird refolding step on the second polypeptide mixture with a thirdrefold buffer comprising about 100 mM to 1M alkali metal salt and/oralkaline metal salt for at least about 30 to 180 minutes; (f)maintaining the MYC-containing polypeptides in the third refold bufferfor a period of time sufficient to produce biologically activenanoparticles having a number average diameter of between about 80 nmand about 150 nm, wherein contacting an anti-CD3 or anti-CD28 activatedT-cell with the biologically active nanoparticles under conditionssuitable for T-cell proliferation, augments one or more of theactivation, survival, or proliferation of the T-cell compared with ananti-CD3 or anti-CD28 activated T-cell that is not contacted with thebiologically active nanoparticles.
 20. The method of claim 19, whereinthe first refolding step, second refolding step, and/or third refoldingstep comprise performing the step by buffer exchange.
 21. The method ofclaim 20, wherein buffer exchange is performed using tangential flowfiltration.
 22. The method of claim 19, wherein the alkali metal saltcomprises one more of a sodium salt, a lithium salt, and a potassiumsalt, wherein the sodium salt comprises one or more of sodium chloride(NaCl), sodium bromide, sodium bisulfate, sodium sulfate, sodiumbicarbonate, or sodium carbonate, wherein the lithium salt comprises oneor more of lithium chloride, lithium bromide, lithium bisulfate, lithiumsulfate, lithium bicarbonate, or lithium carbonate, and wherein thepotassium salt comprises one or more of potassium chloride, potassiumbromide, potassium bisulfate, potassium sulfate, potassium bicarbonate,or potassium carbonate.
 23. (canceled)
 24. The method of claim 19,wherein the alkaline metal salt comprises one more of a magnesium saltand a calcium salt, wherein the magnesium salt comprises one or more ofmagnesium chloride, magnesium bromide, magnesium bisulfate, magnesiumsulfate, magnesium bicarbonate, or magnesium carbonate, and wherein thecalcium salt comprises one or more of calcium chloride, calcium bromide,calcium bisulfate, calcium sulfate, calcium bicarbonate, or calciumcarbonate. 25.-26. (canceled)
 27. The method of claim 19, wherein thealkali metal salt comprises NaCl and the first, second, and/or thirdrefold buffers comprise about 500 mM NaCl.
 28. The method of claim 19,wherein the concentration of denaturing agent in step (a) is from about1 M to about 10 M, wherein the denaturing agent comprises one or more ofguanidine, guanidine hydrochloride, guanidine chloride, guanidinethiocyanate, urea, thiourea, lithium perchlorate, magnesium chloride,phenol, betain, sarcosine, carbamoyl sarcosine, taurine,dimethylsulfoxide (DMSO); alcohols such as propanol, butanol andethanol; detergents, such as sodium dodecyl sulfate (SDS), N-lauroylsarcosine, Zwittergents, non-detergent sulfobetains (NDSB), TRITONX-100, NONIDET™ P-40, the TWEEN™ series and BRIJ™ series; hydroxidessuch as sodium and\or potassium hydroxide.
 29. (canceled)
 30. The methodof claim 19, wherein the first refold buffer, the second refold buffer,and/or third refold buffer each independently comprise a bufferingagent, wherein the buffering agent comprises one or more of TRIS(Tris[hydroxymethyl]aminomethane), HEPPS(N-[2-Hydroxyethyl]piperazine-N′-[3-propane-sulfonic acid]), CAP SO(3-[Cyclohexylamino]-2-hydroxy-1-propanesulfonic acid), AMP(2-Amino-2-methyl-1-propanol), CAPS(3[Cyclohexylamino]-1-propanesulfonic acid), CHES(2[N-Cyclohexylamino]ethanesulfonic acid), arginine, lysine, and sodiumborate.
 31. (canceled)
 32. The method of claim 30, wherein eachbuffering agent is independently present at a concentration from about 1mM to about 1M.
 33. The method of claim 19, wherein the first refoldbuffer, second refold buffer, and/or third refold buffer eachindependently comprise an oxidizing agent and a reducing agent, whereina mole ratio of oxidizing reagent to reducing agent is from about 2:1 toabout 20:1, wherein the oxidizing agent is included in a concentrationfrom about 0.1 mM to about 10 mM, and wherein the oxidizing agent isincluded in a concentration from about 0.1 mM to about 10 mM.
 34. Themethod of claim 33, wherein the oxidizing agent comprises cysteine,glutathione disulfide (“oxidized glutathione”), or both.
 35. (canceled)36. The method of claim 33, wherein the reducing agent comprises one ormore of beta-mercaptoethanol (BME), dithiothreitol (DTT),dithioerythritol (DTE), tris(2-carboxyethyl)phosphine, (TCEP), cystine,cysteamine, thioglycolate, glutathione, and sodium borohydride. 37.(canceled)
 38. The method of claim 19, wherein the denaturing agentcomprises 6-8 M urea.
 39. (canceled)
 40. The method of claim 19, whereinthe first, second, and/or third refold buffers comprise glutathioneand/or oxidized glutathione, wherein the first, second, and/or thirdrefold buffers comprise 5 mM glutathione and/or 1 mM oxidizedglutathione.
 41. (canceled)
 42. The method of claim 19, wherein thefirst, second, and/or third refold buffers comprise glycerol.
 43. Themethod of claim 19, wherein step (f) is performed for at least 5 hours,at least 10 hours, or for 10-12 hours. 44.-45. (canceled)
 46. The methodof claim 19, wherein step (f) further comprises stirring theMYC-containing polypeptides in the third refold buffer at less than 1000rpm.
 47. The method of claim 19, wherein MYC-containing polypeptides arerecombinant polypeptides.
 48. The method of claim 47, wherein the methodfurther comprises isolating a recombinant MYC-containing polypeptidefrom a microbial host cell.
 49. The method of claim 48, wherein themicrobial host cell is E. coli.
 50. The method of claim 48, whereinisolating a recombinant MYC-containing polypeptide from a microbial hostcell comprises expressing the MYC-containing polypeptide from aninducible promoter, or wherein isolating a recombinant MYC-containingpolypeptide from a microbial host cell comprises purifying theMYC-containing polypeptide using affinity chromatography and/or anionexchange chromatography.
 51. (canceled)
 52. The method of claim 19,wherein the MYC-containing polypeptide is acetylated.
 53. The method ofclaim 19, wherein the MYC-containing polypeptide comprises a MYC fusionpeptide, comprising a protein transduction domain linked to a MYCpolypeptide.
 54. The method of claim 53, wherein the MYC fusion peptidefurther comprises one or more molecules that link the proteintransduction domain and the MYC polypeptide.
 55. The method of claim 19,wherein the MYC-containing polypeptide comprises a MYC fusion peptidewith the following general structure: protein transduction domain-X-MYCsequence, wherein -X- is molecule that links the protein transductiondomain and the MYC sequence.
 56. The method of claim 53, wherein theprotein transduction domain sequence is a TAT protein transductiondomain sequence.
 57. The method of claim 56, wherein the TAT proteintransduction domain sequence is selected from the group consisting ofTAT[48-57] and TAT[57-48].
 58. The method of claim 19, wherein theMYC-containing polypeptide is a MYC fusion peptide comprising SEQ ID NO:1 or
 10. 59. The composition of claim 19, wherein the nanoparticles havean average diameter from about 100 nm and about 110 nm.
 60. (canceled)